Genetic polymorphisms sensitively predicting adverse drug reactions (adr) and drug efficacy

ABSTRACT

The invention provides diagnostic methods and kits including oligo and/or polynucleotides or derivatives, including as well antibodies determining whether a human subject is at risk of getting adverse drug reaction after statin therapy or whether the human subject is a high or low responder or a good a or bad metabolizer of statins. The invention provides further diagnostic methods and kits including antibodies determining whether a human subject is at risk for a cardiovascular disease. Still further the invention provides polymorphic sequences and other genes. The present invention further relates to isolated polynucleotides encoding a phenotype associated (PA) gene polypeptide useful in methods to identify therapeutic agents and useful for preparation of a medicament to treat cardiovascular disease or influence drug response, the polynucleotide is selected from the group comprising: SEQ ID 1-21 with allelic variation as indicated in the sequences section contained in a functional surrounding like full length cDNA for PA gene polypeptide and with or without the PA gene promoter sequence.

TECHNICAL FIELD

This invention relates to genetic polymorphisms useful for assessingcardiovascular risks in humans, including, but not limited to,atherosclerosis, ischemia/reperfusion, hypertension, restenosis,arterial inflammation, myocardial infarction, and stroke. In addition itrelates to genetic polymorphisms useful for assessing the response tolipid lowering drug therapy. Specifically, the present inventionidentifies and describes gene variations which are individually presentin humans with cardiovascular disease states, relative to humans withnormal, or non-cardiovascular disease states, and/or in response tomedications relevant to cardiovascular disease. Further, the presentinvention provides methods for the identification and therapeutic use ofcompounds as treatments of cardiovascular disease. Moreover, the presentinvention provides methods for the diagnostic monitoring of patientsundergoing clinical evaluation for the treatment of cardiovasculardisease, and for monitoring the efficacy of compounds in clinicaltrials. Still further, the present invention provides methods to usegene variations to predict personal medication schemes omitting adversedrug reactions and allowing an adjustment of the drug dose to achievemaximum benefit for the patient. Additionally, the present inventiondescribes methods for the diagnostic evaluation and prognosis of variouscardiovascular diseases, and for the identification of subjectsexhibiting a predisposition to such conditions.

BACKGROUND OF THE INVENTION

Cardiovascular disease is a major health risk throughout theindustrialized world.

Cardiovascular diseases include but are not limited by the followingdisorders of the heart and the vascular system: congestive heartfailure, myocardial infarction, atherosclerosis, ischemic diseases ofthe heart, coronary heart disease, all kinds of atrial and ventriculararrhythmias, hypertensive vascular diseases and peripheral vasculardiseases.

Heart failure is defined as a pathophysiologic state in which anabnormality of cardiac function is responsible for the failure of theheart to pump blood at a rate commensurate with the requirement of themetabolizing tissue. It includes all forms of pumping failure such ashigh-output and low-output, acute and chronic, right-sided orleft-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease incoronary blood flow that follows a thrombotic occlusion of a coronaryartery previously narrowed by arteriosclerosis. MI prophylaxis (primaryand secondary prevention) is included as well as the acute treatment ofMI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow isrestricted resulting in an perfusion which is inadequate to meet themyocardial requirement for oxygen. This group of diseases include stableangina, unstable angina and asymptomatic ischemia.

Arrhythmias include all forms of atrial and ventricular tachyarrhythmias(atrial tachycardia, atrial flutter, atrial fibrillation,atrio-ventricular reentrant tachycardia, preexitation syndrome,ventricular tachycardia, ventricular flutter, ventricular fibrillation)as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds ofsecondary arterial hypertension (renal, endocrine, neurogenic, others).

Peripheral vascular diseases are defined as vascular diseases in whicharterial and/or venous flow is reduced resulting in an imbalance betweenblood supply and tissue oxygen demand. It includes chronic peripheralarterial occlusive disease (PAOD), acute arterial thrombosis andembolism, inflammatory vascular disorders, Raynaud's phenomenon andvenous disorders.

Atherosclerosis, the most prevalent of vascular diseases, is theprincipal cause of heart attack, stroke, and gangrene of theextremities, and thereby the principal cause of death. Atherosclerosisis a complex disease involving many cell types and molecular factors(for a detailed review, see Ross, 1993, Nature 362: 801-809 and Lusis,A. J., Nature 407, 233-241 (2000)). The process, in normal circumstancesa protective response to insults to the endothelium and smooth musclecells (SMCs) of the wall of the artery, consists of the formation offibrofatty and fibrous lesions or plaques, preceded and accompanied byinflammation. The advanced lesions of atherosclerosis may occlude theartery concerned, and result from an excessiveinflammatory-fibroproliferative response to numerous different forms ofinsult. For example, shear stresses are thought to be responsible forthe frequent occurrence of atherosclerotic plaques in regions of thecirculatory system where turbulent blood flow occurs, such as branchpoints and irregular structures.

The first observable event in the formation of an atherosclerotic plaqueoccurs when blood-borne monocytes adhere to the vascular endotheliallayer and transmigrate through to the sub-endothelial space. Adjacentendothelial cells at the same time produce oxidized low densitylipoprotein (LDL). These oxidized LDLs are then taken up in largeamounts by the monocytes through scavenger receptors expressed on theirsurfaces. In contrast to the regulated pathway by which native LDL(nLDL) is taken up by nLDL specific receptors, the scavenger pathway ofuptake is not regulated by the monocytes.

These lipid-filled monocytes are called foam cells, and are the majorconstituent of the fatty streak. Interactions between foam cells and theendothelial and SMCs which surround them lead to a state of chroniclocal inflammation which can eventually lead to smooth muscle cellproliferation and migration, and the formation of a fibrous plaque. Suchplaques occlude the blood vessel concerned and thus restrict the flow ofblood, resulting in ischemia.

Ischemia is a condition characterized by a lack of oxygen supply intissues of organs due to inadequate perfusion. Such inadequate perfusioncan have number of natural causes, including atherosclerotic orrestenotic lesions, anemia, or stroke, to name a few. Many medicalinterventions, such as the interruption of the flow of blood duringbypass surgery, for example, also lead to ischemia In addition tosometimes being caused by diseased cardiovascular tissue, ischemia maysometimes affect cardiovascular tissue, such as in ischemic heartdisease. Ischemia may occur in any organ, however, that is suffering alack of oxygen supply.

The most common cause of ischemia in the heart is atheroscleroticdisease of epicardial coronary arteries. By reducing the lumen of thesevessels, atherosclerosis causes an absolute decrease in myocardialperfusion in the basal state or limits appropriate increases inperfusion when the demand for flow is augmented. Coronary blood flow canalso be limited by arterial thrombi, spasm, and, rarely, coronaryemboli, as well as by ostial narrowing due to luetic aortitis.Congenital abnormalities, such as anomalous origin of the left anteriordescending coronary artery from the pulmonary artery, may causemyocardial ischemia and infarction in infancy, but this cause is veryrare in adults. Myocardial ischemia can also occur if myocardial oxygendemands are abnormally increased, as in severe ventricular hypertrophydue to hypertension or aortic stenosis. The latter can be present withangina that is indistinguishable from that caused by coronaryatherosclerosis. A reduction in the oxygen-carrying capacity of theblood, as in extremely severe anemia or in the presence ofcarboxy-hemoglobin, is a rare cause of myocardial ischemia. Notinfrequently, two or more causes of ischemia will coexist, such as anincrease in oxygen demand due to left ventricular hypertrophy and areduction in oxygen supply secondary to coronary atherosclerosis.

The foregoing studies are aimed at defining the role of particular genevariations presumed to be involved in the misleading of normal cellularfunction leading to cardiovascular disease. However, such approachescannot identify the full panoply of gene variations that are involved inthe disease process.

At present, the only available treatments for cardiovascular disordersare pharmaceutical based medications that are not targeted to anindividual's actual defect; examples include angiotensin convertingenzyme (ACE) inhibitors and diuretics for hypertension, insulinsupplementation for non-insulin dependent diabetes mellitus (NIDDM),cholesterol reduction strategies for dyslipidaemia, anticoagulants, βblockers for cardiovascular disorders and weight reduction strategiesfor obesity. If targeted treatment strategies were available it might bepossible to predict the response to a particular regime of therapy andcould markedly increase the effectiveness of such treatment. Althoughtargeted therapy requires accurate diagnostic tests for diseasesusceptibility, once these tests are developed the opportunity toutilize targeted therapy will become widespread. Such diagnostic testscould initially serve to identify individuals at most risk ofhypertension and could allow them to make changes in lifestyle or dietthat would serve as preventative measures. The benefits associated bycoupling the diagnostic tests with a system of targeted therapy couldinclude the reduction in dosage of administered drugs and thus theamount of unpleasant side effects suffered by an individual. In moresevere cases a diagnostic test may suggest that earlier surgicalintervention would be useful in preventing a further deterioration incondition.

It is an object of the invention to provide genetic diagnosis ofpredisposition or susceptibility for cardiovascular diseases. Anotherrelated object is to provide treatment to reduce or prevent or delay theonset of disease in those predisposed or susceptible to this disease. Afurther object is to provide means for carrying out this diagnosis.

Accordingly, a first aspect of the invention provides a method ofdiagnosis of disease in an individual, said method comprisingdetermining one, various or all genotypes in said individual of thegenes listed in the Examples.

In another aspect, the invention provides a method of identifying anindividual predisposed or susceptible to a disease, said methodcomprising determining one, various or all genotypes in said individualof the genes listed in the Examples.

The invention is of advantage in that it enables diagnosis of a diseaseor of certain disease states via genetic analysis which can yielduseable results before onset of disease symptoms, or before onset ofsevere symptoms. The invention is further of advantage in that itenables diagnosis of predisposition or susceptibility to a disease or ofcertain disease states via genetic analysis.

The invention may also be of use in confirming or corroborating theresults of other diagnostic methods. The diagnosis of the invention maythus suitably be used either as an isolated technique or in combinationwith other methods and apparatus for diagnosis, in which latter case theinvention provides a further test on which a diagnosis may be assessed.

The present invention stems from using allelic association as a methodfor genotyping individuals; allowing the investigation of the moleculargenetic basis for cardiovascular diseases. In a specific embodiment theinvention tests for the polymorphisms in the sequences of the listedgenes in the Examples. The invention demonstrates a link between thispolymorphisms and predispositions to cardiovascular diseases by showingthat allele frequencies significantly differ when individuals with “bad”serum lipids are compared to individuals with “good” serum levels. Themeaning of “good and bad” serum lipid levels is defined in Table 1a.

The PROCAM algorithm defines also a risk assessment based on lipids(LDL-cholesterol, HDL-cholesterol, triglycerides) and risk factors likesmoking, high blood pressure or diabetes mellitus (Assmann, G., Schulte,H. von Eckardstein, A; Am J Cardiol 77 (1996): 1179-1184).

Certain disease states would benefit, that is to say the suffering ofthe patient may be reduced or prevented or delayed, by administration oftreatment or therapy in advance of disease appearance; this can be morereliably carried out if advance diagnosis of predisposition orsusceptibility to disease can be diagnosed.

Pharmacogenomics and Adverse Drug Reactions

Adverse drug reactions (ADRs) remain a major clinical problem. A recentmetaanalysis suggested that in the USA in 1994, ADRs were responsiblefor 100 000 deaths, making them between the fourth and sixth commonestcause of death (Lazarou 1998, J. Am. Med. Assoc. 279:1200). Althoughthese figures have been heavily criticized, they emphasize theimportance of ADRs. Indeed, there is good evidence that ADRs account for5% of all hospital admissions and increase the length of stay inhospital by two days at an increased cost of ˜$2500 per patient. ADRsare also one of the commonest causes of drug withdrawal, which hasenormous financial implications for the pharmaceutical industry. ADRs,perhaps fortunately, only affect a minority of those taking a particulardrug. Although factors that determine susceptibility are unclear in mostcases, there is increasing interest in the role of genetic factors.Indeed, the role of inheritable variations in predisposing patients toADRs has been appreciated since the late 1950s and early 1960s throughthe discovery of deficiencies in enzymes such as pseudocholinesterase(butyrylcholinesterase) and glucose-6-phosphate dehydrogenase (G6PD).More recently, with the first draft of the human genome just completed,there has been renewed interest in this area with the introduction ofterms such as pharmacogenomics and toxicogenomics. Essentially, the aimof pharmacogenomics is to produce personalized medicines, wherebyadministration of the drug class and dosage is tailored to an individualgenotype. Thus, the term pharmacogenomics embraces both efficacy andtoxicity.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors(“statins”) specifically inhibit the enzyme HMG-CoA reductase whichcatalyzes the rate limiting step in cholesterol biosynthesis. Thesedrugs are effective in reducing the primary and secondary risk ofcoronary artery disease and coronary events, such as heart attack, inmiddle-aged and older men and women, in both diabetic and non-diabeticpatients, and are often prescribed for patients with hyperlipidemia.Statins used in secondary prevention of coronary artery or heart diseasesignificantly reduce the risk of stroke, total mortality and morbidityand attacks of myocardial ischemia; the use of statins is alsoassociated with improvements in endothelial and fibrinolytic functionsand decreased platelet thrombus formation.

The tolerability of these drugs during long term administration is animportant issue. Adverse reactions involving skeletal muscle are notuncommon, and sometimes serious adverse reactions involving skeletalmuscle such as myopathy and rhabdomyolysis may occur, requiringdiscontinuation of the drug. In addition an increase in serum creatinekinase (CK) may be a sign of a statin related adverse event. The extendof such adverse events can be read from the extend of the CK levelincrease (as compared to the upper normal limit [ULN]).

Occasionally arhralgia, alone or in association with myalgia, has beenreported. Also an elevation of liver transaminases has been associatedwith statin administration.

It was shown that the drug response to statin therapy is a classeffects, i.e. all known and presumably also all so far undiscoveredstatins share the same benefical and harmful effects (Ucar, M. et al.,Drug Safety 2000, 22:441). It follows that the discovery of diagnostictools to predict the drug response to a single statin will also be ofaid to guide therapy with other statins.

The present invention provides diagnostic tests to predict the patient'sindividual response to statin therapy. Such responses include, but arenot limited by the extent of adverse drug reactions, the level of lipidlowering or the drug's influence on disease states. Those diagnostictests may predict the response to statin therapy either alone or incombination with another diagnostic test or another drug regimen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part on the discovery that aspecific allele of a polymorphic region of a so called “candidate gene”(as defined below) is associated with CVD or drug response.

For the present invention the following candidate genes were analyzed:

Genes found to be expressed in cardiac tissue (Hwang et al., Circulation1997, 96:4146-4203).

Genes from the following metabolic pathways and their regulatoryelements:

Lipid Metabolism

Numerous studies have shown a connection between serum lipid levels andcardiovascular diseases. Candidate genes falling into this group includebut are not limited by genes of the cholesterol pathway, apolipoproteinsand their modifiying factors.

Coagulation

Ischemic diseases of the heart and in particular myocardial infarctionmay be caused by a thrombotic occlusion. Genes falling into this groupinclude all genes of the coagulation cascade and their regulatoryelements.

Inflammation

Complications of atherosclerosis are the most common causes of death inWestern societies. In broad outline atherosclerosis can be considered tobe a form of chronic inflammation resulting from interaction modifiedlipoproteins, monocyte-derived macrophages, T cells, and the normalcellular elements of the arterial wall. This inflammatory process canultimately lead to the development of complex lesions, or plaques, thatprotrude into the arterial lumen. Finally plaque rupture and thrombosisresult in the acute clinical complications of myocardial infarction andstroke (Glass et al., Cell 2001, 104:503-516).

It follows that all genes related to inflammatory processes, includingbut not limited by cytokines, cytokine receptors and cell adhesionmolecules are candidate genes for CVD.

Glucose and Energy Metabolism

As glucose and energy metabolism is interdependent with the metabolismof lipids (see above) also the former pathways contain candidate genes.Energy metabolism in general also relates to obesity, which is anindependent risk factor for CVD (Melanson et al., Cardiol Rev 20019:202-207). In addition high blood glucose levels are associated withmany microvascular and macrovascular complications and may thereforeaffect an individuals disposition to CVD (Duckworth, Curr AtherosclerRep 2001, 3:383-391).

Hypertension

As hypertension is an independent risk factor for CVD, also genes thatare involved in the regulation of systolic and diastolic blood pressureaffect an individuals risk for CVD (Safar, Curr Opin Cardiol 2000,15:258-263). Interestingly hypertension and diabetes (see above) appearto be interdependent, since hypertension is approximately twice asfrequent in patients with diabetes compared with patients without thedisease. Conversely, recent data suggest that hypertensive persons aremore predisposed to the development of diabetes than are normotensivepersons (Sowers et al., Hypertension 2001, 37:1053-1059).

Genes Related to Drug Response

Those genes include metabolic pathways involved in the absorption,distribution, metabolism, excretion and toxicity (ADMET) of drugs.Prominent members of this group are the cytochrome P450 proteins whichcatalyze many reactions involved in drug metabolism.

Unclassified Genes

As stated above, the mechanisms that lead to cardiovascular diseases ordefine the patient's individual response to drugs are not completelyelucidated. Hence also candidate genes were analysed, which could not beassigned to the above listed categories. The present invention is basedat least in part on the discovery of polymorphisms, that lie in genomicregions of unknown physiological function.

Results

After conducting an association study, we surprisingly found polymorphicsites in a number of candidate genes which show a strong correlationwith the following phenotypes of the patients analysed: “Healthy” asused herein refers to individuals that neither suffer from existing CVD,nor exhibit an increased risk for CVD through their serum lipid levelprofile. “CVD prone” as used herein refers to individuals with existingCVD and/or a serum lipid profile that confers a high risk to get CVD(see Table 1a for definitions of healthy and CVD prone serum lipidlevels). “High responder” as used herein refers to patients who benefitfrom relatively small amounts of a given drug. “Low responder” as usedherein refers to patients who need relatively high doses in order toobtain benefit from the medication. “Tolerant patient” refers toindividuals who can tolerate high doses of a medicament withoutexhibiting adverse drug reactions. “ADR patient” as used herein refersto individuals who suffer from ADR or show clinical symptoms (likecreatine kinase elevation in blood) even after receiving only minordoses of a medicament (see Table 1b for a detailed-definition of drugresponse phenotypes).

Polymorphic sites in candidate genes that were found to be significantlyassociated with either of the above mentioned phenotypes will bereferred to as “phenotype associated SNPs” (PA SNPs). The respectivegenomic loci that harbour PA SNPs will be referred to as “phenotypeassociated genes” (PA genes), irrespective of the actual function ofthis gene locus.

In particular we surprisingly found PA SNPs associated with CVD, drugefficacy (EFF) or adverse drug reactions (ADR) in the following genes.

ABCA1: ATP-Binding Cassette, Sub-Family A (ABC1), Member 1

The membrane-associated protein encoded by this gene is a member of thesuperfamily of ATP-binding cassette (ABC) transporters. ABC proteinstransport various molecules across extra- and intracellular membranes.ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP,MRP, ALD, OABP, GCN20, White). This protein is a member of the ABC1subfamily. Members of the ABC1 subfamily comprise the only major ABCsubfamily found exclusively in multicellular eukaryotes. Withcholesterol as its substrate, this protein functions as a cholesteralefflux pump in the cellular lipid removal pathway. Mutations in thisgene have been associated with Tangier's disease and familialhigh-density lipoprotein deficiency.

ABCB1: ATP-Binding Cassette, Sub-Family B (MDR/TAP), Member 1

The membrane-associated protein encoded by this gene is a member of thesuperfamily of ATP-binding cassette (ABC) transporters. ABC proteinstransport various molecules across extra- and intra-cellular membranes.ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP,MRP, ALD, OABP, GCN20, White). This protein is a member of the MDR/TAPsubfamily. Members of the MDR/TAP subfamily are involved in multidrugresistance. The protein encoded by this gene is an ATP-dependent drugefflux pump for xenobiotic compounds with broad substrate specificity.It is responsible for decreased drug accumulation in multidrug-resistantcells and often mediates the development of resistance to anticancerdrugs. This protein also functions as a transporter in the blood-brainbarrier.

ABCG2: ATP-Binding Cassette, Sub-Family G (WHITE), Member 2

The membrane-associated protein encoded by this gene is included in thesuperfamily of ATP-binding cassette (ABC) transporters. ABC proteinstransport various molecules across extra- and intra-cellular membranes.ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP,MRP, ALD, OABP, GCN20, White). This protein is a member of the Whitesubfamily. Alternatively referred to as a breast cancer resistanceprotein, this protein functions as a xenobiotic transporter which mayplay a major role in multi drug resistance. It likely serves as acellular defense mechanism in response to mitoxantrone and anthracyclineexposure. Significant expression of this protein has been observed inthe placenta, which may suggest a potential role for this molecule inplacenta tissue.

CD69: CD69 Antigen (p60, Early T-Cell Activation Antigen)

Member of the Ca2+-dependent (C-type) lectin superfamily of type IItransmembrane receptors; acts as signal transducing receptor, involvedin lymphocyte proliferation.

CSF3: Colony Stimulating Factor 3 (Granulocyte)

Granulocyte colony stimulating factor 3.

Hexokinase II

Hexokinases phosphorylate glucose to produce glucose-6-phosphate, thuscommitting glucose to the glycolytic pathway. This gene encodeshexokinase 2, the predominant form found in skeletal muscle. Itlocalizes to the outer membrane of mitochondria. Expression of this geneis insulin-responsive, and studies in rat suggest that it is involved inthe increased rate of glycolysis seen in rapidly growing cancer cells.

Human bcl-2 mRNA

BCL2 is an integral inner mitochondrial membrane protein that blocks theapoptotic death of some cells such as lymphocytes. Constitutiveexpression of BCL2, such as in the case of translocation of BCL2 to Igheavy chain locus, is thought to be the cause of follicular lymphoma.Two transcript variants, produced by alternate splicing, differ in theirC-terminal ends. Variant 1 is represented by a 5.5 kb mRNA and includesmost of exon 1 and exon 2. A 3.5 kb mRNA represents variant 2; itincludes all of exon 1 and lacks exon 2 sequence.

Human Methylenetetrahydrofolate Dehydrogenase-MethenyltetrahydrofolateCyclohydrolase-Formyltetrahydrofolate Synthetase mRNA, Complete cds.

This gene encodes a protein that possesses three distinct enzymaticactivities, 5,10-methylenetetrahydrofolate dehydrogenase,5,10-methenyltetrahydrofolate cyclohydrolase and10-formyltetrahydrofolate synthetase. Each of these activities catalyzesone of three sequential reactions in the interconversion of 1-carbonderivatives of tetrahydrofolate, which are substrates for methionine,thymidylate, and de novo purine syntheses. The trifunctional enzymaticactivities are conferred by two major domains, an aminoterminal portioncontaining the dehydrogenase and cyclohydrolase activities and a largersynthetase domain.

Human Thermostable Phenol Sulfotransferase (STP2) Gene

Phenol-metabolizing sulfotransferase 2; sulfonates simple planarphenols.

IFIT2: Interferon-Induced Protein with Tetratricopeptide Repeats 2(ISG-54K)

IL4: Interleukin 4

Interleukin 4; cytokine that stimulates the proliferation of T cells.

ITGA9: Integrin, Alpha 9

Alpha 9 subunit of integrin; may be involved in cell-cell andcell-matrix interactions; member of a family of cell-surface proteins.

KIAA0229: KIAA0229 Protein

MMP1: Matrix Metalloproteinase 1 (Interstitial Collagenase)

Proteins of the matrix metalloproteinase (MMP) family are involved inthe breakdown of extracellular matrix in normal physiological processes,such as embryonic development, reproduction, and tissue remodeling, aswell as in disease processes, such as arthritis and metastasis. MostMMP's are secreted as inactive proproteins which are activated whencleaved by extracellular proteinases. This gene encodes a secretedenzyme which breaks down the interstitial collagens, types I, II, andIII. The gene is part of a cluster of MMP genes which localize tochromosome 11q22.3.

MMP16: Matrix Metalloproteinase 16 (Membrane-Inserted)

Proteins of the matrix metalloproteinase (MMP) family are involved inthe breakdown of extracellular matrix in normal physiological processes,such as embryonic development, reproduction, and tissue remodeling, aswell as in disease processes, such as arthritis and metastasis. MostMMP's are secreted as inactive proproteins which are activated whencleaved by extracellular proteinases. This gene produces twotranscripts, which encode a membrane-bound form and a soluble form ofthe protein. Both forms of the protein activate MMP2 by cleavage. Thisgene was once referred to as MT-MMP2, but was renamed as MT-MMP3 orMMP16.

OXA1Hs

Involved in the assembly of complexes of the electron transport chain.

PROCR: Protein C Receptor, Endothelial (EPCR)

Endothelial Protein C receptor; binds protein C in a calcium-dependentmanner; member of the CD1/major histocompatibility complex superfamily.

SERPINE1: Serine (or Cysteine) Proteinase Inhibitor, Clade E (Nexin,Plasminogen Activator Inhibitor Type 1), Member 1

Plasminogen activator inhibitor I; regulates fibrinolysis; member of theserpin family of serine protease inhibitors.

STX1A: Syntaxin 1A (Brain)

Syntaxin 1A (brain); involved in intracellular transport andneurotransmitter release. As SNPs are linked to other SNPs inneighboring genes on a chromosome (Linkage Disequilibrium) those SNPscould also be used as marker SNPs. In a recent publication it was shownthat SNPs are linked over 100 kb in some cases more than 150 kb (ReichD. E. et al. Nature 411, 199-204, 2001). Hence SNPs lying in regionsneighbouring PA SNPs could be linked to the latter and by this being adiagnostic marker. These associations could be performed as describedfor the gene polymorphism in methods.

Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.Moreover, the definitions by itself are intended to explain a furtherbackground of the invention.

The term “allele”, which is used interchangeably herein with “allelicvariant” refers to alternative forms of a gene or portions thereof.Alleles occupy the same locus or position on homologous chromosomes.When a subject has two identical alleles of a gene, the subject is saidto be homozygous for the gene or allele. When a subject has twodifferent alleles of a gene, the subject is said to be heterozygous forthe gene. Alleles of a specific gene can differ from each other in asingle nucleotide, or several nucleotides, and can includesubstitutions, deletions, and insertions of nucleotides. An allele of agene can also be a form of a gene containing a mutation.

The term “allelic variant of a polymorphic region of a gene” refers to aregion of a gene having one of several nucleotide sequences found inthat region of the gene in other individuals.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present invention.

The term “a homologue of a nucleic acid” refers to a nucleic acid havinga nucleotide sequence having a certain degree of homology with thenucleotide sequence of the nucleic acid or complement thereof. Ahomologue of a double stranded nucleic acid having SEQ ID NO. X isintended to include nucleic acids having a nucleotide sequence which hasa certain degree of homology with SEQ ID NO. X or with the complementthereof. Preferred homologous of nucleic acids are capable ofhybridizing to the nucleic acid or complement thereof.

The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a hybridization assay.

The term interact is also meant to include “binding” interactionsbetween molecules. Interactions may be, for example, protein-protein,protein-nucleic acid, protein-small molecule or small molecule-nucleicacid in nature.

The term “intronic sequence” or “intronic nucleotide sequence” refers tothe nucleotide sequence of an intron or portion thereof.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.

Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.

The term “lipid” shall refer to a fat or fat-like substance that isinsoluble in polar solvents such as water. The term “lipid” is intendedto include true fats (e.g. esters of fatty acids and glycerol); lipids(phospholipids, cerebrosides, waxes); sterols (cholesterol, ergosterol)and lipoproteins (e.g. HDL, LDL and VLDL).

The term “locus” refers to a specific position in a chromosome. Forexample, a locus of a gene refers to the chromosomal position of thegene.

The term “modulation” as used herein refers to both up-regulation,(i.e., activation or stimulation), for example by agonizing, anddown-regulation (i.e. inhibition or suppression), for example byantagonizing of a bioactivity (e.g. expression of a gene).

The term “molecular structure” of a gene or a portion thereof refers tothe structure as defined by the nucleotide content (including deletions,substitutions, additions of one or more nucleotides), the nucleotidesequence, the state of methylation, and/or any other modification of thegene or portion thereof.

The term “mutated gene” refers to an allelic form of a gene, which iscapable of altering the phenotype of a subject having the mutated generelative to a subject which does not have the mutated gene. If a subjectmust be homozygous for this mutation to have an altered phenotype, themutation is said to be recessive. If one copy of the mutated gene issufficient to alter the genotype of the subject, the mutation is said tobe dominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous (for that gene) subject, the mutation is said to beco-dominant.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,derivatives, variants and analogs of either RNA or DNA made fromnucleotide analogs, including peptide nucleic acids (PNA), morpholinooligonucleotides (J. Summerton and D. Weller, Antisense and Nucleic AcidDrug Development 7:187 (1997)) and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotides. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes ofclarity, when referring herein to a nucleotide of a nucleic acid, whichcan be DNA or an RNA, the term “adenosine”, “cytidine”, “guanosine”, and“thymidine” are used. It is understood that if the nucleic acid is RNA,a nucleotide having a uracil base is uridine.

The term “nucleotide sequence complementary to the nucleotide sequenceset forth in SEQ ID NO. x” refers to the nucleotide sequence of thecomplementary strand of a nucleic acid strand having SEQ ID NO. x. Theterm “complementary strand” is used herein interchangeably with the term“complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double stranded nucleic acids, the complement of anucleic acid having SEQ ID NO. x refers to the complementary strand ofthe strand having SEQ ID NO. x or to any nucleic acid having thenucleotide sequence of the complementary strand of SEQ ID NO. x. Whenreferring to a single stranded nucleic acid having the nucleotidesequence SEQ ID NO. x, the complement of this nucleic acid is a nucleicacid having a nucleotide sequence which is complementary to that of SEQID NO. x. The nucleotide sequences and complementary sequences thereofare always given in the 5′ to 3′ direction. The term “complement” and“reverse complement” are used interchangeably herein.

The term “operably linked” is intended to mean that the promoter isassociated with the nucleic acid in such a manner as to facilitatetranscription of the nucleic acid.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion thereof. A portion of a gene of which there are atleast two different forms, i.e., two different nucleotide sequences, isreferred to as a “polymorphic region of a gene”. A polymorphic regioncan be a single nucleotide, the identity of which differs in differentalleles. A polymorphic region can also be several nucleotides long.

A “polymorphic gene” refers to a gene having at least one polymorphicregion.

To describe a “polymorphic site” in a nucleotide sequence often there isused an “ambiguity code” that stands for the possible variations ofnucleotides in one site. The list of ambiguity codes is summarized inthe following table: Ambiguity Codes (IUPAC Nomenclature) B c/g/t Da/g/t H a/c/t K g/t M a/c N a/c/g/t R a/g S c/g V a/c/g W a/t Y c/t

So, for example, a “R” in a nucleotide sequence means that either an “a”or a “g” could be at that position.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

A “regulatory element”, also termed herein “regulatory sequence isintended to include elements which are capable of modulatingtranscription from a basic promoter and include elements such asenhancers and silencers. The term “enhancer”, also referred to herein as“enhancer element”, is intended to include regulatory elements capableof increasing, stimulating, or enhancing transcription from a basicpromoter. The term “silencer”, also referred to herein as “silencerelement” is intended to include regulatory elements capable ofdecreasing, inhibiting, or repressing transcription from a basicpromoter. Regulatory elements are typically present in 5′ flankingregions of genes. However, regulatory elements have also been shown tobe present in other regions of a gene, in particular in introns. Thus,it is possible that genes have regulatory elements located in introns,exons, coding regions, and 3′ flanking sequences. Such regulatoryelements are also intended to be encompassed by the present inventionand can be identified by any of the assays that can be used to identifyregulatory elements in 5′ flanking regions of genes.

The term “regulatory element” further encompasses “tissue specific”regulatory elements, i.e., regulatory elements which effect expressionof the selected DNA sequence preferentially in specific cells (e.g.,cells of a specific tissue). gene expression occurs preferentially in aspecific cell if expression in this cell type is significantly higherthan expression in other cell types. The term “regulatory element” alsoencompasses non-tissue specific regulatory elements, i.e., regulatoryelements which are active in most cell types. Furthermore, a regulatoryelement can be a constitutive regulatory element, i.e., a regulatoryelement which constitutively regulates transcription, as opposed to aregulatory element which is inducible, i.e., a regulatory element whichis active primarily in response to a stimulus. A stimulus can be, e.g.,a molecule, such as a hormone, cytokine, heavy metal, phorbol ester,cyclic AMP (cAMP), or retinoic acid.

Regulatory elements are typically bound by proteins, e.g., transcriptionfactors. The term “transcription factor” is intended to include proteinsor modified forms thereof, which interact preferentially with specificnucleic acid sequences, i.e., regulatory elements, and which inappropriate conditions stimulate or repress transcription. Sometranscription factors are active when they are in the form of a monomer.Alternatively, other transcription factors are active in the form of adimer consisting of two identical proteins or different proteins(heterodimer). Modified forms of transcription factors are intended torefer to transcription factors having a post-translational modification,such as the attachment of a phosphate group. The activity of atranscription factor is frequently modulated by a post-translationalmodification. For example, certain transcription factors are active onlyif they are phosphorylated on specific residues. Alternatively,transcription factors can be active in the absence of phosphorylatedresidues and become inactivated by phosphorylation. A list of knowntranscription factors and their DNA binding site can be found, e.g., inpublic databases, e.g., TFMATRIX Transcription Factor Binding SiteProfile database.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule of theinvention to hybridize to at least approximately 6, 12, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130 or 140 consecutive nucleotides ofeither strand of a gene.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

“Adverse drug reaction” (ADR) as used herein refers to an appreciablyharmful or unpleasant reaction, resulting from an intervention relatedto the use of a medicinal product, which predicts hazard from futureadministration and warrants prevention or specific treatment, oralteration of the dosage regimen, or withdrawal of the product. In it'smost severe form an ADR might lead to the death of an individual.

The term “Drug Response” is intended to mean any response that a patientexhibits upon drug administration. Specifically drug response includesbeneficial, i.e. desired drug effects, ADR or no detectable reaction atall. More specifically the term drug response could also have aqualitative meaning, i.e. it embraces low or high beneficial effects,respectively and mild or severe ADR, respectively. The term “StatinResponse” as used herein refers to drug response after statinadministration. An individual drug response includes also a good or badmetabolizing of the drug, meaning that “bad metabolizers” accumulate thedrug in the body and by this could show side effects of the drug due toaccumulative overdoses.

“Candidate gene” as used herein includes genes that can be assigned toeither normal cardiovascular function or to metabolic pathways that arerelated to onset and/or progression of cardiovascular diseases.

With regard to drug response the term “candidate gene” includes genesthat can be assigned to distinct phenotypes regarding the patient'sresponse to drug administration. Those phenotypes may include patientswho benefit from relatively small amounts of a given drug (highresponders) or patients who need relatively high doses in order toobtain the same benefit (low responders). In addition those phenotypesmay include patients who can tolerate high doses of a medicament withoutexhibiting ADR, or patients who suffer from ADR even after receivingonly low doses of a medicament.

As neither the development of cardiovascular diseases nor the patient'sresponse to drug administration is completely understood, the term“candidate gene” may also comprise genes with presently unknownfunction.

“PA SNP” (phenotype associated SNP) refers to a polymorphic site whichshows a significant association with a patients phenotype healthy,diseased, low or high responder, drug tolerant, ADR prone, etc.)

“PA gene” (phenotype associated gene) refers to a genomic locusharbouring a PA SNP, irrespective of the actual function of this genelocus.

PA gene polypeptide refers to a polypeptide encoded at least in part bya PA gene.

The term “Haplotype” as used herein refers to a group of two or moreSNPs that are functionally and/or spatially linked. I.e. haplotypesdefine groups of SNPs that lie inside genes belonging to identical (orrelated metabolic) pathways and/or lie on the same chromosome.Haplotypes are expected to give better predictive/diagnostic informationthan a single SNP

The term “statin” is intended to embrace all inhibitors of the enzyme3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Statinsspecifically inhibit the enzyme HMG-CoA reductase which catalyzes therate limiting step in cholesterol biosynthesis. Known statins areAtorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Pravastatin andSimvastatin.

Methods for Assessing Cardiovascular Status

The present invention provides diagnostic methods for assessingcardiovascular status in a human individual. Cardiovascular status asused herein refers to the physiological status of an individual'scardiovascular system as reflected in one or more markers or indicators.Status markers include without limitation clinical measurements such as,e.g., blood pressure, electrocardiographic profile, and differentiatedblood flow analysis as well as measurements of LDL- and HDL-Cholesterollevels, other lipids and other well established clinical parameters thatare standard in the art. Status markers according to the inventioninclude diagnoses of one or more cardiovascular syndromes, such as,e.g., hypertension, acute myocardial infarction, silent myocardialinfarction, stroke, and atherosclerosis. It will be understood that adiagnosis of a cardiovascular syndrome made by a medical practitionerencompasses clinical measurements and medical judgement. Status markersaccording to the invention are assessed using conventional methods wellknown in the art. Also included in the evaluation of cardiovascularstatus are quantitative or qualitative changes in status markers withtime, such as would be used, e.g., in the determination of anindividual's response to a particular therapeutic regimen.

The methods are carried out by the steps of:

-   (i) determining the sequence of one or more polymorphic positions    within one, several or all of the genes listed in Examples or other    genes mentioned in this file in the individual to establish a    polymorphic pattern for the individual; and-   (ii) comparing the polymorphic pattern established in (i) with the    polymorphic patterns of humans exhibiting different markers of    cardiovascular status. The polymorphic pattern of the individual is,    preferably, highly similar and, most preferably, identical to the    polymorphic pattern of individuals who exhibit particular status    markers, cardiovascular syndromes, and/or particular patterns of    response to therapeutic interventions. Polymorphic patterns may also    include polymorphic positions in other genes which are shown, in    combination with one or more polymorphic positions in the genes    listed in the Examples, to correlate with the presence of particular    status markers. In one embodiment, the method involves comparing an    individual's polymorphic pattern with polymorphic patterns of    individuals who have been shown to respond positively or negatively    to a particular therapeutic regimen. Therapeutic regimen as used    herein refers to treatments aimed at the elimination or amelioration    of symptoms and events associated cardiovascular disease. Such    treatments include without limitation one or more of alteration in    diet, lifestyle, and exercise regimen; invasive and noninvasive    surgical techniques such as atherectomy, angioplasty, and coronary    bypass surgery; and pharmaceutical interventions, such as    administration of ACE inhibitors, angiotensin II receptor    antagonists, diuretics, alpha-adrenoreceptor antagonists, cardiac    glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor    antagonists, calcium channel blockers, HMG-CoA reductase inhibitors,    imidazoline receptor blockers, endothelin receptor blockers, organic    nitrites, and modulators of protein function of genes listed in the    Examples. Interventions with pharmaceutical agents not yet known    whose activity correlates with particular polymorphic patterns    associated with cardiovascular disease are also encompassed. It is    contemplated, for example, that patients who are candidates for a    particular therapeutic regimen will be screened for polymorphic    patterns that correlate with responsivity to that particular    regimen.

In a preferred embodiment, the method involves comparing an individual'spolymorphic pattern with polymorphic patterns of individuals who exhibitor have exhibited one or more markers of cardiovascular disease, suchas, e.g., elevated LDL-Cholesterol levels, high blood pressure, abnormalelectrocardiographic profile, myocardial infarction, stroke, oratherosclerosis.

In another embodiement, the method involves comparing an individual'spolymorphic pattern with polymorphic patterns of individuals who exhibitor have exhibited one or more drug related phenotypes, such as, e.g.,low or high drug response, or adverse drug reactions.

In practicing the methods of the invention, an individual's polymorphicpattern can be established by obtaining DNA from the individual anddetermining the sequence at predetermined polymorphic positions in thegenes such as those described in this file.

The DNA may be obtained from any cell source. Non-limiting examples ofcell sources available in clinical practice include blood cells, buccalcells, cervicovaginal cells, epithelial cells from urine, fetal cells,or any cells present in tissue obtained by biopsy. Cells may also beobtained from body fluids, including without limitation blood, saliva,sweat, urine, cerebrospinal fluid, feces, and tissue exudates at thesite of infection or inflammation. DNA is extracted from the cell sourceor body fluid using any of the numerous methods that are standard in theart. It will be understood that the particular method used to extractDNA will depend on the nature of the source.

Diagnostic and Prognostic Assays

The present invention provides methods for determining the molecularstructure of at least one polymorphic region of a gene, specific allelicvariants of said polymorphic region being associated with cardiovasculardisease. In one embodiment, determining the molecular structure of apolymorphic region of a gene comprises determining the identity of theallelic variant. A polymorphic region of a gene, of which specificalleles are associated with cardiovascular disease can be located in anexon, an intron, at an intron/exon border, or in the promoter of thegene.

The invention provides methods for determining whether a subject has, oris at risk, of developing a cardiovascular disease. Such disorders canbe associated with an aberrant gene activity, e.g., abnormal binding toa form of a lipid, or an aberrant gene protein level. An aberrant geneprotein level can result from an aberrant transcription orpost-transcriptional regulation. Thus, allelic differences in specificregions of a gene can result in differences of gene protein due todifferences in regulation of expression. In particular, some of theidentified polymorphisms in the human gene may be associated withdifferences in the level of transcription, RNA maturation, splicing, ortranslation of the gene or transcription product.

In preferred embodiments, the methods of the invention can becharacterized as comprising detecting, in a sample of cells from thesubject, the presence or absence of a specific allelic variant of one ormore polymorphic regions of a gene. The allelic differences can be: (i)a difference in the identity of at least one nucleotide or (ii) adifference in the number of nucleotides, which difference can be asingle nucleotide or several nucleotides.

A preferred detection method is allele specific hybridization usingprobes overlapping the polymorphic site and having about 5, 10, 20, 25,or 30 nucleotides around the polymorphic region. Examples of probes fordetecting specific allelic variants of the polymorphic region located inintron X are probes comprising a nucleotide sequence set forth in any ofSEQ ID NO. X. In a preferred embodiment of the invention, several probescapable of hybridizing specifically to allelic variants are attached toa solid phase support, e.g., a “chip”. Oligonucleotides can be bound toa solid support by a variety of processes, including lithography. Forexample a chip can hold up to 250,000 oligonucleotides (GeneChip,Affymetrix). Mutation detection analysis using these chips comprisingoligonucleotides, also termed “DNA probe arrays” is described e.g., inCronin et al. (1996) Human Mutation 7:244 and in Kozal et al. (1996)Nature Medicine 2:753. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment. For example, theidentity of the allelic variant of the nucleotide polymorphism ofnucleotide A or G at position 33 of Seq ID 1 (baySNP179) and that ofother possible polymorphic regions can be determined in a singlehybridization experiment.

In other detection methods, it is necessary to first amplify at least aportion of a gene prior to identifying the allelic variant.Amplification can be performed, e.g., by PCR and/or LCR, according tomethods known in the art. In one embodiment, genomic DNA of a cell isexposed to two PCR primers and amplification for a number of cyclessufficient to produce the required amount of amplified DNA. In preferredembodiments, the primers are located between 40 and 350 base pairsapart. Preferred primers for amplifying gene fragments of genes of thisfile are listed in Table 2 in the Examples.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment, any of a variety of sequencing reactions known in theart can be used to directly sequence at least a portion of a gene anddetect allelic variants, e.g., mutations, by comparing the sequence ofthe sample sequence with the corresponding wild-type (control) sequence.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxam and Gilbert (Proc. Natl. Acad Sci USA (1977) 74:560)or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463). It is alsocontemplated that any of a variety of automated sequencing proceduresmay be utilized when performing the subject assays (Biotechniques (1995)19:448), including sequencing by mass spectrometry (see, for example,U.S. Pat. No. 5,547,835 and international patent application PublicationNumber WO 94/16101, entitled DNA Sequencing by Mass Spectrometry by H.Koster; U.S. Pat. No. 5,547,835 and international patent applicationPublication Number WO 94121822 entitled “DNA Sequencing by MassSpectrometry Via Exonuclease Degradation” by H. Koster), and U.S. Pat.No. 5,605,798 and International Patent Application No. PCT/US96/03651entitled DNA Diagnostics Based on Mass Spectrometry by H. Koster; Cohenet al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) ApplBiochem Biotechnol 38:147-159). It will be evident to one skilled in theart that, for certain embodiments, the occurrence of only one, two orthree of the nucleic acid bases need be determined in the sequencingreaction. For instance, A-track or the like, e.g., where only onenucleotide is detected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No.5,580,732 entitled “Method of DNA sequencing employing a mixedDNA-polymer chain probe” and U.S. Pat. No. 5,571,676 entitled “Methodfor mismatch-directed in vitro DNA sequencing”.

In some cases, the presence of a specific allele of a gene in DNA from asubject can be shown by restriction enzyme analysis. For example, aspecific nucleotide polymorphism can result in a nucleotide sequencecomprising a restriction site which is absent from the nucleotidesequence of another allelic variant.

In other embodiments, alterations in electrophoretic mobility is used toidentify the type of gene allelic variant. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control nucleicacids are denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In anotherpreferred embodiment, the subject method utilizes heteroduplex analysisto separate double stranded heteroduplex molecules on the basis ofchanges in electrophoretic mobility (Keen et al. (1991) Trends Genet7:5).

In yet another embodiment, the identity of an allelic variant of apolymorphic region is obtained by analyzing the movement of a nucleicacid comprising the polymorphic region in polyacrylamide gels containinga gradient of denaturant is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al (1985) Nature 313:495). When DGGE isused as the method of analysis, DNA will be modified to insure that itdoes not completely denature, for example by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

Examples of techniques for detecting differences of at least onenucleotide between 2 nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl.Acad. Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the simultaneous detection of several nucleotide changesin different polymorphic regions of gene. For example, oligonucleotideshaving nucleotide sequences of specific allelic variants are attached toa hybridizing membrane and this membrane is then hybridized with labeledsample nucleic acid. Analysis of the hybridization signal will thenreveal the identity of the nucleotides of the sample nucleic acid.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used. Oligonucleotides used asprimers for specific amplification may carry the allelic variant ofinterest in the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238; Newton et al. (1989) Nucl.Acids Res. 17:2503). This technique is also termed “PROBE” for ProbeOligo Base Extension. In addition it may be desirable to introduce anovel restriction site in the region of the mutation to createcleavage-based detection (Gasparini et al (1992) Mol. Cell Probes 6:1).

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science241:1077-1080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g, biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect specific allelic variants of a polymorphic region of agene. For example, U.S. Pat. No. 5,593,826 discloses an OLA using anoligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each LA reactioncan be detected by using hapten specific antibodies that are labeledwith different enzyme reporters, alkaline phosphatase or horseradishperoxidase. This system permits the detection of the two alleles using ahigh throughput format that leads to the production of two differentcolors.

The invention further provides methods for detecting single nucleotidepolymorphisms in a gene. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each patient.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA TM isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990),Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA TM in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For determining the identity of the allelic variant of a polymorphicregion located in the coding region of a gene, yet other methods thanthose described above can be used. For example, identification of anallelic variant which encodes a mutated gene protein can be performed byusing an antibody specifically recognizing the mutant protein in, e.g.,immunohistochemistry or immunoprecipitation. Antibodies to wild-typegene protein are described, e.g., in Acton et al. (1999) Science 271:518(anti-mouse gene antibody cross-reactive with human gene). Otherantibodies to wild-type gene or mutated forms of gene proteins can beprepared according to methods known in the art. Alternatively, one canalso measure an activity of an gene protein, such as binding to a lipidor lipoprotein. Binding assays are known in the art and involve, e.g.,obtaining cells from a subject, and performing binding experiments witha labeled lipid, to determine whether binding to the mutated form of thereceptor differs from binding to the wild-type of the receptor.

If a polymorphic region is located in an exon, either in a coding ornon-coding region of the gene, the identity of the allelic variant canbe determined by determining the molecular structure of the mRNA,pre-mRNA, or cDNA. The molecular structure can be determined using anyof the above described methods for determining the molecular structureof the genomic DNA, e.g., sequencing and SSCP.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits, such as those described above, comprisingat least one probe or primer nucleic acid described herein, which may beconveniently used, e.g., to determine whether a subject has or is atrisk of developing a disease associated with a specific gene allelicvariant.

Sample nucleic acid for using in the above-described diagnostic andprognostic methods can be obtained from any cell type or tissue of asubject. For example, a subject's bodily fluid (e.g. blood) can beobtained by known techniques (e.g. venipuncture) or from human tissueslike heart (biopsies, transplanted organs). Alternatively, nucleic acidtests can be performed on dry samples (e.g. hair or skin). Fetal nucleicacid samples for prenatal diagnostics can be obtained from maternalblood as described in International Patent Application No. WO91/07660 toBianchi. Alternatively, amniocytes or chorionic villi may be obtainedfor performing prenatal testing.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, New York).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

In practicing the present invention, the distribution of polymorphicpatterns in a large number of individuals exhibiting particular markersof cardiovascular status or drug response is determined by any of themethods described above, and compared with the distribution ofpolymorphic patterns in patients that have been matched for age, ethnicorigin, and/or any other statistically or medically relevant parameters,who exhibit quantitatively or qualitatively different status markers.Correlations are achieved using any method known in the art, includingnominal logistic regression, chi square tests or standard least squaresregression analysis. In this manner, it is possible to establishstatistically significant correlations between particular polymorphicpatterns and particular cardiovascular statuses (given in p values). Itis further possible to establish statistically significant correlationsbetween particular polymorphic patterns and changes in cardiovascularstatus or drug response such as, would result, e.g., from particulartreatment regimens. In this manner, it is possible to correlatepolymorphic patterns with responsivity to particular treatments.

In another embodiment of the present invention two or more polymorphicregions are combined to define so called ‘haplotypes’. Haplotypes aregroups of two or more SNPs that are functionally and/or spatiallylinked. It is possible to combine SNPs that are disclosed in the presentinvention either with each other or with additional polymorphic regionsto form a haplotype. Haplotypes are expected to give betterpredictive/diagnostic information than a single SNP.

In a preferred embodiment of the present invention a panel ofSNPs/haplotypes is defined that predicts the risk for CVD or drugresponse. This predictive panel is then used for genotyping of patientson a platform that can genotype multiple SNPs at the same time(Multiplexing). Preferred platforms are e.g. gene chips (Affymetrix) orthe Luminex LabMAP reader. The subsequent identification and evaluationof a patient's haplotype can then help to guide specific andindividualized therapy.

For example the present invention can identify patients exhibitinggenetic polymorphisms or haplotypes which indicate an increased risk foradverse drug reactions. In that case the drug dose should be lowered ina way that the risk for ADR is diminished. Also if the patient'sresponse to drug administration is particularly high (or the patient isbadly metabolizing the drug), the drug dose should be lowered to avoidthe risk of ADR.

In turn if the patient's response to drug administration is low (or thepatient is a particularly high metabolizer of the drug), and there is noevident risk of ADR, the drug dose should be raised to an efficaciouslevel.

It is self evident that the ability to predict a patient's individualdrug response should affect the formulation of a drug, i.e. drugformulations should be tailored in a way that they suit the differentpatient classes (low/high responder, poor/good metabolizer, ADR pronepatients). Those different drug formulations may encompass differentdoses of the drug, i.e. the medicinal products contains low or highamounts of the active substance. In another embodiement of the inventionthe drug formulation may contain additional substances that facilitatethe beneficial effects and/or diminish the risk for ADR (Folkers et al.1991, U.S. Pat. No. 5,316,765).

Isolated Polymorphic Nucleic Acids, Probes, and Vectors

The present invention provides isolated nucleic acids comprising thepolymorphic positions described herein for human genes; vectorscomprising the nucleic acids; and transformed host cells comprising thevectors. The invention also provides probes which are useful fordetecting these polymorphisms.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA, are used. Suchtechniques are well known and are explained fully in, for example,Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glovered.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Nucleic AcidHybridization, 1985, (Hames and Higgins); Ausubel et al., CurrentProtocols in Molecular Biology, 1997, (John Wiley and Sons); and Methodsin Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds.,respectively).

Insertion of nucleic acids (typically DNAs) comprising the sequences ina functional surrounding like full length cDNA of the present inventioninto a vector is easily accomplished when the termini of both the DNAsand the vector comprise compatible restriction sites. If this cannot bedone, it may be necessary to modify the termini of the DNAs and/orvector by digesting back single-stranded DNA overhangs generated byrestriction endonuclease cleavage to produce blunt ends, or to achievethe same result by filling in the single-stranded termini with anappropriate DNA polymerase.

Alternatively, any site desired may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., 1988,Science 239:48. The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

The nucleic acids may be isolated directly from cells or may bechemically synthesized using known methods. Alternatively, thepolymerase chain reaction (PCR) method can be used to produce thenucleic acids of the invention, using either chemically synthesizedstrands or genomic material as templates. Primers used for PCR can besynthesized using the sequence information provided herein and canfurther be designed to introduce appropriate new restriction sites, ifdesirable, to facilitate incorporation into a given vector forrecombinant expression.

The nucleic acids of the present invention may be flanked by native genesequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-noncoding regions, andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates,morpholines etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.). Nucleic acids may contain one or moreadditional covalently linked moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine,etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g.,metals, radioactive metals, iron, oxidative metals, etc.), andalkylators. PNAs are also included. The nucleic acid may be derivatizedby formation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the nucleic acid sequences of thepresent invention may also be modified with a label capable of providinga detectable signal, either directly or indirectly. Exemplary labelsinclude radioisotopes; fluorescent molecules, biotin, and the like.

The invention also provides nucleic acid vectors comprising the genesequences or derivatives or fragments thereof of genes described in theExamles. A large number of vectors, including plasmid and fungalvectors, have been described for replication and/or expression in avariety of eukaryotic and prokaryotic hosts, and may be used for genetherapy as well as for simple cloning or protein expression.Non-limiting examples of suitable vectors include without limitation pUCplasmids, pET plasmids (Novagen, Inc., Madison, Wis.), or pRSET or pREP(Invitrogen, San Diego, Calif.), and many appropriate host cells, usingmethods disclosed or cited herein or otherwise known to those skilled inthe relevant art. The particular choice of vector/host is not criticalto the practice of the invention.

Suitable host cells may be transformed/transfected/infected asappropriate by any suitable method including electroporation, CaCl₂mediated DNA uptake, fungal or viral infection, microinjection,microprojectile, or other established methods. Appropriate host cellsincluded bacteria, archebacteria, fungi, especially yeast, and plant andanimal cells, especially mammalian cells. A large number oftranscription initiation and termination regulatory regions have beenisolated and shown to be effective in the transcription and translationof heterologous proteins in the various hosts. Examples of theseregions, methods of isolation, manner of manipulation, etc. are known inthe art. Under appropriate expression conditions, host cells can be usedas a source of recombinantly produced peptides and polypeptides encodedby genes of the Examples. Nucleic acids encoding peptides orpolypeptides from gene sequences of the Examples may also be introducedinto cells by recombination events. For example, such a sequence can beintroduced into a cell and thereby effect homologous recombination atthe site of an endogenous gene or a sequence with substantial identityto the gene. Other recombination-based methods such as non-homologousrecombinations or deletion of endogenous genes by homologousrecombination may also be used.

In case of proteins that form heterodimers or other multimers, both orall subunits have to be expressed in one system or cell.

The nucleic acids of the present invention find use as probes for thedetection of genetic polymorphisms and as templates for the recombinantproduction of normal or variant peptides or polypeptides encoded bygenes listed in the Examples.

Probes in accordance with the present invention comprise withoutlimitation isolated nucleic acids of about 10-100 bp, preferably 15-75bp and most preferably 17-25 bp in length, which hybridize at highstringency to one or more of the polymorphic sequences disclosed hereinor to a sequence immediately adjacent to a polymorphic position.Furthermore, in some embodiments a full-length gene sequence may be usedas a probe. In one series of embodiments, the probes span thepolymorphic positions in genes disclosed herein. In another series ofembodiments, the probes correspond to sequences immediately adjacent tothe polymorphic positions.

Polymorphic Polypeptides and Polymorphism-Specific Antibodies

The present invention encompasses isolated peptides and polypeptidesencoded by genes listed in the Examples comprising polymorphic positionsdisclosed herein. In one preferred embodiment, the peptides andpolypeptides are useful screening targets to identify cardiovasculardrugs. In another preferred embodiments, the peptides and polypeptidesare capable of eliciting antibodies in a suitable host animal that reactspecifically with a polypeptide comprising the polymorphic position anddistinguish it from other polypeptides having a different sequence atthat position.

Polypeptides according to the invention are preferably at least five ormore residues in length, preferably at least fifteen residues. Methodsfor obtaining these polypeptides are described below. Many conventionaltechniques in protein biochemistry and immunology are used. Suchtechniques are well known and are explained in Immunochemical Methods inCell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press,London); Scopes, 1987, Protein Purification: Principles and Practice,Second Edition (Springer-Verlag, N.Y.) and Handbook of ExperimentalImmunology, 1986, Volumes I-IV (Weir and Blackwell eds.).

Nucleic acids comprising protein-coding sequences can be used to directthe ITT recombinant expression of polypeptides encoded by genesdisclosed herein in intact cells or in cell-free translation systems.The known genetic code, tailored if desired for more efficientexpression in a given host organism, can be used to synthesizeoligonucleotides encoding the desired amino acid sequences. Thepolypeptides may be isolated from human cells, or from heterologousorganisms or cells (including, but not limited to, bacteria, fungi,insect, plant, and mammalian cells) into which an appropriateprotein-coding sequence has been introduced and expressed. Furthermore,the polypeptides may be part of recombinant fusion proteins.

Peptides and polypeptides may be chemically synthesized by commerciallyavailable automated procedures, including, without limitation, exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. The polypeptides arepreferably prepared by solid phase peptide synthesis as described byMerrifield, 1963, J. Am. Chem. Soc. 85:2149.

Methods for polypeptide purification are well-known in the art,including, without limitation, preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase BPLC, gel filtration, ionexchange and partition chromatography, and counter-current distribution.For some purposes, it is preferable to produce the polypeptide in arecombinant system in which the protein contains an additional sequencetag that facilitates purification, such as, but not limited to, apolyhistidine sequence. The polypeptide can then be purified from acrude lysate of the host cell by chromatography on an appropriatesolid-phase matrix. Alternatively, antibodies produced against peptidesencoded by genes disclosed herein, can be used as purification reagents.Other purification methods are possible.

The present invention also encompasses derivatives and homologues of thepolypeptides. For some purposes, nucleic acid sequences encoding thepeptides may be altered by substitutions, additions, or deletions thatprovide for functionally equivalent molecules, i.e.,function-conservative variants. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofsimilar properties, such as, for example, positively charged amino acids(arginine, lysine, and histidine); negatively charged amino acids(aspartate and glutamate); polar neutral amino acids; and non-polaramino acids.

The isolated polypeptides may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.They may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

The present invention also encompasses antibodies that specificallyrecognize the polymorphic positions of the invention and distinguish apeptide or polypeptide containing a particular polymorphism from onethat contains a different sequence at that position. Such polymorphicposition-specific antibodies according to the present invention includepolyclonal and monoclonal antibodies. The antibodies may be elicited inan animal host by immunization with peptides encoded by genes disclosedherein or may be formed by in vitro immunization of immune cells. Theimmunogenic components used to elicit the antibodies may be isolatedfrom human cells or produced in recombinant systems. The antibodies mayalso be produced in recombinant systems programmed with appropriateantibody-encoding DNA. Alternatively, the antibodies may be constructedby biochemical reconstitution of purified heavy and light chains. Theantibodies include hybrid antibodies (i.e., containing two sets of heavychain/light chain combinations, each of which recognizes a differentantigen), chimeric antibodies (i.e., in which either the heavy chains,light chains, or both, are fusion proteins), and univalent antibodies(i.e., comprised of a heavy chain/light chain complex bound to theconstant region of a second heavy chain). Also included are Fabfragments, including Fab′ and F(ab).sub.2 fragments of antibodies.Methods for the production of all of the above types of antibodies andderivatives are well-known in the art and are discussed in more detailbelow. For example, techniques for producing and processing polyclonalantisera are disclosed in Mayer and Walker, 1987, Immunochemical Methodsin Cell and Molecular Biology, (Academic Press, London). The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal antibody-producing cell lines can be created by cellfusion, and also by other techniques such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., Schreier et al., 1980, Hybridoma Techniques; U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,44,887; 4,466,917; 4,472,500;4,491,632; and 4,493,890. Panels of monoclonal antibodies producedagainst peptides encoded by genes disclosed herein can be screened forvarious properties; i.e. for isotype, epitope affinity, etc.

The antibodies of this invention can be purified by standard methods,including but not limited to preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ionexchange and partition chromatography, and countercurrent distribution.Purification methods for antibodies are disclosed, e.g., in The Art ofAntibody Purification, 1989, Amicon Division, W. R. Grace & Co. Generalprotein purification methods are described in Protein Purification:Principles and Practice, R. K. Scopes, Ed., 1987, Springer-Verlag, NewYork, N.Y.

Methods for determining the immunogenic capability of the disclosedsequences and the characteristics of the resulting sequence-specificantibodies and immune cells are well-known in the art. For example,antibodies elicited in response to a peptide comprising a particularpolymorphic sequence can be tested for their ability to specificallyrecognize that polymorphic sequence, i.e., to bind differentially to apeptide or polypeptide comprising the polymorphic sequence and thusdistinguish it from a similar peptide or polypeptide containing adifferent sequence at the same position.

Kits

As set forth herein, the invention provides diagnostic methods, e.g.,for determining the identity of the allelic variants of polymorphicregions present in the gene loci of genes disclosed herein, whereinspecific allelic variants of the polymorphic region are associated withcardiovascular diseases. In a preferred embodiment, the diagnostic kitcan be used to determine whether a subject is at risk of developing acardiovascular disease. This information could then be used, e.g., tooptimize treatment of such individuals.

In preferred embodiments, the kit comprises a probe or primer which iscapable of hybridizing to a gene and thereby identifying whether thegene contains an allelic variant of a polymorphic region which isassociated with a risk for cardiovascular disease. The kit preferablyfurther comprises instructions for use in diagnosing a subject ashaving, or having a predisposition, towards developing a cardiovasculardisease. The probe or primers of the kit can be any of the probes orprimers described in this file.

Preferred kits for amplifying a region of a gene comprising apolymorphic region of interest comprise one, two or more primers.

Antibody-Based Diagnostic Methods and Kits:

The invention also provides antibody-based methods for detectingpolymorphic patterns in a biological sample. The methods comprise thesteps of: (i) contacting a sample with one or more antibodypreparations, wherein each of the antibody preparations is specific fora particular polymorphic form of the proteins encoded by genes disclosedherein, under conditions in which a stable antigen-antibody complex canform between the antibody and antigenic components in the sample; and(ii) detecting any antigen-antibody complex formed in step (i) using anysuitable means known in the art, wherein the detection of a complexindicates the presence of the particular polymorphic form in the sample.

Typically, immunoassays use either a labelled antibody or a labelledantigenic component (e.g., that competes with the antigen in the samplefor binding to the antibody). Suitable labels include without imitationenzyme-based, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays that amplify the signals from the probe are alsoknown, such as, for example, those that utilize biotin and avidin, andenzyme-labelled immunoassays, such as ELISA assays.

The present invention also provides kits suitable for antibody-baseddiagnostic applications. Diagnostic kits typically include one or moreof the following components:

-   (i) Polymorphism-specific antibodies. The antibodies may be    pre-labelled; alternatively, the antibody may be unlabelled and the    ingredients for labelling may be included in the kit in separate    containers, or a secondary, labelled antibody is provided; and-   (ii) Reaction components: The kit may also contain other suitably    packaged reagents and materials needed for the particular    immunoassay protocol, including solid-phase matrices, if applicable,    and standards.

The kits referred to above may include instructions for conducting thetest. Furthermore, in preferred embodiments, the diagnostic kits areadaptable to high-throughput and/or automated operation.

Drug Targets and Screening Methods

According to the present invention, nucleotide sequences derived fromgenes disclosed herein and peptide sequences encoded by genes disclosedherein, particularly those that contain one or more polymorphicsequences, comprise useful targets to identify cardiovascular drugs,i.e., compounds that are effective in treating one or more clinicalsymptoms of cardiovascular disease. Furthermore, especially when aprotein is a multimeric protein that are build of two or more subunits,is a combination of different polymorphic subunits very useful.

Drug targets include without limitation (i) isolated nucleic acidsderived from the genes disclosed herein, and (ii) isolated peptides andpolypeptides encoded by genes disclosed herein, each of which comprisesone or more polymorphic positions.

In Vitro Screening Methods:

In one series of embodiments, an isolated nucleic acid comprising one ormore polymorphic positions is tested in vitro for its ability to bindtest compounds in a sequence-specific manner. The methods comprise:

-   (i) providing a first nucleic acid containing a particular sequence    at a polymorphic position and a second nucleic acid whose sequence    is identical to that of the first nucleic acid except for a    different sequence at the same polymorphic position;-   (ii) contacting the nucleic acids with a multiplicity of test    compounds under conditions appropriate for binding; and-   (iii) identifying those compounds that bind selectively to either    the first or second nucleic acid sequence.

Selective binding as used herein refers to any measurable difference inany parameter of binding, such as, e.g., binding affinity, bindingcapacity, etc.

In another series of embodiments, an isolated peptide or polypeptidecomprising one or more polymorphic positions is tested in vitro for itsability to bind test compounds in a sequence-specific manner. Thescreening methods involve:

-   (i) providing a first peptide or polypeptide containing a particular    sequence at a polymorphic position and a second peptide or    polypeptide whose sequence is identical to the first peptide or    polypeptide except for a different sequence at the same polymorphic    position;-   (ii) contacting the polypeptides with a multiplicity of test    compounds under conditions appropriate for binding; and-   (iii) identifying those compounds that bind selectively to one of    the nucleic acid sequences.

In preferred embodiments, high-throughput screening protocols are usedto survey a large number of test compounds for their ability to bind thegenes or peptides disclosed above in a sequence-specific manner.

Test compounds are screened from large libraries of synthetic or naturalcompounds. Numerous means are currently used for random and directedsynthesis of saccharide, peptide, and nucleic acid based compounds.Synthetic compound libraries are commercially available from MaybridgeChemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.),Brandon Associates (Merrimack, N.H.), and Microsource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from e.g. PanLaboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readilyproducible. Additionally, natural and synthetically produced librariesand compounds are readily modified through conventional chemical,physical, and biochemical means.

In Vivo Screening Methods:

Intact cells or whole animals expressing polymorphic variants of genesdisclosed herein can be used in screening methods to identify candidatecardiovascular drugs.

In one series of embodiments, a permanent cell line is established froman individual exhibiting a particular polymorphic pattern.Alternatively, cells (including without limitation mammalian, insect,yeast, or bacterial cells) are programmed to express a gene comprisingone or more polymorphic sequences by introduction of appropriate DNA.Identification of candidate compounds can be achieved using any suitableassay, including without limitation (i) assays that measure selectivebinding of test compounds to particular polymorphic variants of proteinsencoded by genes disclosed herein; (ii) assays that measure the abilityof a test compound to modify (i.e., inhibit or enhance) a measurableactivity or function of proteins encoded by genes disclosed herein; and(iii) assays that measure the ability of a compound to modify (i.e.,inhibit or enhance) the transcriptional activity of sequences derivedfrom the promoter (i.e., regulatory) regions of genes disclosed herein.

In another series of embodiments, transgenic animals are created inwhich (i) one or more human genes disclosed herein, having differentsequences at particular polymorphic positions are stably inserted intothe genome of the transgenic animal; and/or (ii) the endogenous genesdisclosed herein are inactivated and replaced with human genes disclosedherein, having different sequences at particular polymorphic positions.See, e.g., Coffman, Semin. Nephrol. 17:404, 1997; Esther et al., Lab.Invest. 74:953, 1996; Murakami et al., Blood Press. Suppl. 2:36, 1996.Such animals can be treated with candidate compounds and monitored forone or more clinical markers of cardiovascular status.

The following are intended as non-limiting examples of the invention.

Material and Methods

Genotyping of patient DNA with the Pyrosequencing™ Method as describedin the patent application WO 9813523:

First a PCR is set up to amplify the flanking regions around a SNP.Therefor 2 ng of genomic DNA (patient sample) are mixed with a primerset(20-40 pmol) producing a 75 to 320 bp PCR fragment with 0,3 to 1 UQiagens Hot Star Taq Polymerase™ in a total volume of 20 μL. One primeris biotinylated depending on the direction of the sequencing primer. Toforce the biotinylated prixmer to be incorporated it is used 0,8 fold.

For primer design, programms like Oligo 6™ (Molecular Biology Insights)or Primer Select™ (DNAStar) are used. PCR setup is performed by aBioRobot 3000™ from Qiagen. PCR takes place in T1 or TgradientThermocyclers™ from Biometra.

The whole PCR reaction is transferred into a PSQ plate™ (Pyrosequencing)and prepared using the Sample Prep Tool™ and SNP Reagent Kit™ fromPyrosequencing according to their instructions.

Preparation of Template for Pyrosequencing™:

Sample Preparation Using PSQ 96 Sample Prep Tool:

-   1. Mount the PSQ 96 Sample Prep Tool Cover onto the PSQ 96 Sample    Prep Tool as follows: Place the cover on the desk, retract the 4    attachment rods by separating the handle from the magnetic rod    holder, fit the magnetic rods into the holes of the cover plate,    push the handle downward until a click is heard. The PSQ 96 Sample.    Prep Tool is now ready for use.-   2. To transfer beads from one plate to another, place the covered    tool into the PSQ 96 Plate containing the samples and lower the    magnetic rods by separating the handle from the magnetic rod holder.    Move the tool up and down a few times then wait for 30-60 seconds.    Transfer the beads into a new PSQ 96 plate containing the solution    of choice.-   3. Release the beads by lifting the magnetic rod holder, bringing it    together with the handle. Move the tool up and down a few times to    make sure that the beads are released.

All steps are performed at room temperature unless otherwise stated.

Immobilization of PCR Product:

Biotinylated PCR products are immobilized on streptavidin-coatedDynabeads™ M-280 Streptavidin. Parallel immobilization of severalsamples are performed in the PSQ 96 Plate.

-   1. Mix PCR product, 20 μl of a well optimized PCR, with 25 μl    2×BW-buffer II. Add 60-150 μg Dynabeads. It is also possible to add    a mix of Dynabeads and 2×BW-buffer II to the PCR product yielding a    final BW-buffer II concentration of approximately 1x.-   2. Incubate at 65° C. for 15 min agitation constantly to keep the    beads dispersed. For optimal immobilization of fragments longer than    300 bp use 30 min incubation time.    Strand Separation:-   3. For strand separation, use the PSQ 96 Sample Prep Tool to    transfer the beads with the immobilized sample to a PSQ 96 Plate    containing 50 μl 0.50 M NaOH per well. Release the beads.-   4. After approximately 1 min, transfer the beads with the    immobilized strand to a PSQ 96 Plate containing 99 μl 1× Annealing    buffer per well and mix thoroughly.-   5. Transfer the beads to a PSQ 96 Plate containing 45 μl of a mix of    1× Annealing buffer and 3-15 pmoles sequencing primer per well.-   6. Heat at 80° C. for 2 minutes in the PSQ 96 Sample Prep.    Thermoplate and move to room temperature.-   7. After reaching room temperature, continue with the sequencing    reaction.    Sequencing Reaction:-   1. Choose the method to be used (“SNP Method”) and enter relevant    information in the PSQ 96 Instrument Control software.-   2. Place the cartridge and PSQ 96 Plate in the PSQ 96 Instrument.-   3. Start the run.    Genotyping Using the ABI 7700/7900 Instrument (TaqMan)

SNP genotypisation using the TaqMan (Applied Biosystems/Perkin Elmer)was performed according to the manufacturer's instructions. The TaqManassay is discussed by Lee et al., Nucleic Acids Research 1993, 21:3761-3766.

Genotyping with a Service Contractor:

Qiagen Genomics, formerly Rapigene, is a service contractor forgenotyping SNPs in patient samples. Their method is based on a primerextension method where two complementary primers are designed for eachgenotype that are labeled with different tags. Depending on the genotypeonly one primer will be elongated together with a certain tag. This tagcan be detected with mass spectrometry and is a measure for therespective genotype. The method is described in the following patent:“Detection and identification of nucleic acid molecules—using tags whichmay be detected by non-fluorescent spectrometry or potentiometry” (WO9727325).

EXAMPLES

To exemplify the present invention and it's utility baySNP 2214 will beused in the following:

baySNP 2214 is an A to T polymorphism and presumably resides in the geneof the human ISG-54K gene (interferon stimulated gene) encoding a 54 kDAprotein (information taken from table 3). baySNP 2214 was genotyped invarious patient cohorts using the primers from table 2. As a result thefollowing number of patients carrying different genotypes were found(information combined from tables 3 and 5a): Geno Geno- Geno- type 11type 12 type 22 baySNP Cohort Total “AA” “AT” “TT” 2214 HELD_MAL_CASE 143 9 2 2214 HELD_MAL_CTRL 18 0 11 7

When comparing the number of male patients exhibiting CVD(HELD_MAL_CASE) with the control cohort (HELD_μL_CTRL) it appears thatthe number of CVD patients carrying the AA genotype is increased,whereas the number of CVD patients carrying the TT genotype isdiminished. This points to higher risk of CVD among male individualswith the CC genotype. Applying statistical tests on those findings thefollowing p-values were obtained (data taken from table 5b): GTYPE GTYPEGTYPE BAYSNP COMPARISON CPVAL XPVAL LRPVAL 2214 HELD_MAL_CC 0.06190.0799 0.0334

As at least one of the GTYPE p values is below 0,05 the association ofgenotype and CVD is regarded as statistically significant. I.e. theanalysis of a patient's genotype can predict the risk to suffer fromCVD. In more detail one can calculate the relative risk to suffer fromCVD when carrying a certain genotype (data taken from table 6a): BAYSNPCOMPARISON GTYPE1 GTYPE2 GTYPE3 RR1 RR2 RR3 2214 HELD_MAL_CC AA AT TT2.64 1.08 0.43

In case of baySNP 2214 the risk to suffer from CVD is 2,64 times higherwhen carrying the AA genotype. This indicates that a AA polymorphism inbaySNP 2214 is an independent risk factor for CVD. On the other handcarriers of a TT genotype have a reduced risk to suffer from CVD.

In addition statistical associations can be calculated on the basis onalleles. This calculation would identify risk alleles instead of riskgenotypes.

In case of baySNP 900073 the following allele counts were obtained (datacombined from tables 3 and 5a): Allele 1 Allele 2 baySNP Cohort Total“C” “G” 900073 CVD_MAL_CASE 69 47 91 900073 CVD_MAL_CTRL 32 12 52

When comparing the number of male patients with CVD (CVD_MAL_CASE) withthe control cohort (CVD_MAL_CTRL) it appears that the number of CVDpatients carrying the C allele is increased, whereas the number of CVDpatients carrying the G allele is diminished. This points to a higherrisk for CVD among male individuals with the C allele. When applyingstatistical tests on those findings the following p-values were obtained(data taken from table 5b): ALLELE ALLELE ALLELE BAYSNP COMPARISON CPVALXPVAL LRPVAL 900073 CVD_MAL 0.026 0.0306 0.0224

As at least one of the ALLELE p values is below 0,05 the association ofallele and CVD risk is regarded as statistically significant (in thisexample significant p values were obtained from all three statisticaltests). I.e. also the analysis of a patient's alleles from baySNP 900073can predict the risk to suffer from CVD. In more detail one cancalculate the relative risk to suffer from CVD when carrying a certainallele (data taken from table 6b): baySNP Allele 1 Allele 2 COMPARISONRR1 RR2 900073 C G CVD_MAL 1.25 0.8

In case of baySNP 900073 the risk to suffer from CVD for males is 1,25times higher when carrying the C allele. This indicates that the Callele of baySNP900073 is an independent risk factor for CVD in males.

Another example is baySNP 4564, which is taken to exemplifypolymorphisms relevant for adverse drug reactions. baySNP 4564 was foundsignificant when comparing male patients with advanced ADR to therespective controls (as defined in table 1b).

The relative risk ratios for the genotypes AA, AG and GG were as follows(data taken from table 6a): BAYSNP COMPARISON GTYPE1 GTYPE2 GTYPE3 RR1RR2 RR3 4564 HELD_MAL_ADR3ULN AA AG GG 0.49 2.05 null

In this case male patients carrying the AG genotype have a 2,05 timeshigher risk to suffer from ADR. In other words those patients shouldeither receive lower doses of statins or switch to an alternativetherapy in order to avoid ADR. On the other hand male patients with theAA genotype appear to be more resistant to ADR and hence better toleratestatin therapy.

As a last example baySNP 8589 is taken: In that case the risk to exhibita high responder phenotype is 1,31 times higher when carrying the Callele. This indicates that the C allele of baySNP 8589 is anindependent risk factor for a high statin response in females. In otherwords those patients should receive lower doses of statins in order toavoid ADR. However due to their ‘high responder’ phenotype they willstill benefit from the drug. In turn carriers of the T allele shouldreceive higher drug doses in order to experience a benefical therapeuticeffect TABLE 1a Definition of “good” and “bad” serum lipid levels “Good”“Bad” LDL-Cholesterol [mg/dL] 125-150 170-200 Cholesterol [mg/dL]190-240 265-315 HDL-Cholesterol [mg/dL]  60-105 30-55 Triglycerides[mg/dL]  45-115 170-450

According to the PROCAM algorithm (Assmann, G., Schulte, H. vonEckardstein, A; Am J Cardiol 77 (1996): 1179-1184) it is possible todefine other cohorts. For example a lipid-based equation would calculatey as follows:y=−0.0146*LDL+0.0418*HDL-0.3362*In(TRIGLY)

Good or bad cohorts could then be defined in the following way(FEM=female, MAL=male):

FEM_GOOD y>=1.4

FEM_BAD y<1.4

MAL_GOOD y>=1.7

MAL_BAD y<1.7 TABLE 1b Definition of drug response phenotypes Lowresponder Decrease of serum LDL of at least 10% and at most 50% uponadministration of 0.8 mg Cerivastatin (female patients) High responderDecrease of serum LDL of at least 50% upon administration of 0.4 mgCerivastatin (female patients) Very low responder Decrease of serum LDLof at least 10% and at most 35% upon administration of 0.8 mgCerivastatin (female patients) Very high responder Decrease of serum LDLof at least 55% upon administration of 0.4 mg Cerivastatin (femalepatients) Ultra low responder Decrease of serum LDL of at least 10% andat most 25% upon administration of 0.8 mg Cerivastatin (female patients)Ultra high responder Decrease of serum LDL of at least 60% uponadministration of 0.4 mg Cerivastatin (female patients) Tolerant patientNo diagnosis of muscle cramps, muscle pain, muscle weakness, myalgia ormyopathy AND serum CK levels below 70 mg/dl in women and below 80 mg/dlin men. ADR patient Diagnosis of muscle cramps, muscle (CK increase atleast 2 × ULN) pain, muscle weakness, myalgia or myopathy OR serum CKlevels higher than 140 mg/dl in women and 160 mg/dl in men. Advanced ADRpatient [ADR3] Serum CK levels higher than 210 mg/dl (advanced CKincrease, at least 3 × ULN)* in women and 240 mg/dl in men Severe ADRpatient [ADR5] Serum CK levels higher than 350 mg/dl (severe CKincrease, at least 5 × ULN)* in women and 400 mg/dl in men*When assembling the cohorts for advanced and severe ADR we focused onthe CK serum levels as those provide a more independent measure ofstatin related ADR.

TABLE 1c Definition of “high” and “low” serum HDL cholesterol levelsMale Female individuals individuals ,High′ HDL-Cholesterol[mg/dL] >=80 >=104 ,Low′ HDL-Cholesterol [mg/dL] <=35 <=37

An informed consent was signed by the patients and control people. Bloodwas taken by a physician according to medical standard procedures.

Samples were collected anonymous and labeled with a patient number. DNAwas extracted using kits from Qiagen. TABLE 2a Oligonucleotide primersused for genotyping using mass spectrometry The baySNP number refers toan internal numbering of the PA SNPs. Primer sequences are listed forpreamplification of the genomic fragments (primers EF and ER) and forsubsequent allele specific PCR of the SNP. baySNP SNP Name Sequence 160C195T ER gacgatgccttcagcacaCTTGGCTTGGAATAGAGA 160 C195T EFGACTATGCGGAGAAGATG 160 C195T CF gggacggtcggtagatCTGAGCTGTGAGAGGGGC 160C195T TF gctggctcggtcaagaCTGAGCTGTGAGAGGGGT 1278 A168G AFgggacggtcggtagatGCCGGCCAGAGCAAGCTA 1278 A168G EF CACTACAGATAGAGGGGTG1278 A168G ER GACGATGCCTTCAGCACAATTGAGATGACAGGTTGAG 1278 A168G GFgctggctcggtcaagaGCCGGCCAGAGCAAGCTG 1371 C507T CRgggacggtcggtagatGGCTCGGGACGATGGGAG 1371 C507T EFGACGATGCCTTCAGCACAGCCCACTCCTACCACAAG 1371 C507T ER GGGGACAGAGAGAAGCAA1371 C507T TR gctggctcggtcaagaGGCTCGGGACGATGGGAA 1806 A201G AFgggacggtcggtagatTGGGCGTCCTGGTGGGCA 1806 A201G EF TCTTCGGGCTAACTCTTT 1806A201G ER GACGATGCCTTCAGCACACTGTCACTCCAAACCTTCT 1806 A201G GFgctggctcggtcaagaTGGGCGTCCTGGTGGGCG 2178 C719T CRgggacggtcggtagatTGGCAAACACGTTCCAGG 2178 C719T EFGACGATGCCTTCAGCACAAGGAAATAGAAGGGGAGGA 2178 C719T ER CCTGTGAACTGCCTGAAC2178 C719T TR gctggctcggtcaagaTGGCAAACACGTTCCAGA 2198 C548T CFgggacggtcggtagatGACAAATGCTTATGAAAC 2198 C548T EF GAAATAACTAGGCGTGGA 2198C548T ER GACGATGCCTTCAGCACATGGGGAAAAATAACAAAG 2198 C548T TFgctggctcggtcaagaGACAAATGCTTATGAAAT 2214 A231T ARgggacggtcggtagatTTGGCTGCACTGCGAAGT 2214 A231T EFGACGATGCCTTCAGCACAAGGAAGGTGAAGGAGAGA 2214 A231T ER GTAGGCATTGTTTGGTATG2214 A231T TR gctggctcggtcaagaTTGGCTGCACTGCGAAGA 2267 C490T CRgggacggtcggtagatATTCTGGGCACCACAGCCG 2267 C490T EFGACGATGCCTTCAGCACACTTCTGAGTGGGCGTTATTAC 2267 C490T ERGGTGGCCAAGGTCGTGCTG 2267 C490T TR gctggctcggtcaagaATTCTGGGCACCACAGCCA3907 A194C ER gacgatgccttcagcacaAGTGGAGAGAGGATGTTTAG 3907 A194C EFGTCTTATGTAGACGCTTGG 3907 A194C AF gggacggtcggtagatTCCCCAGGGCGGGTAAGA3907 A194C CF gctggctcggtcaagaTCCCCAGGGCGGGTAAGC 4564 A446G EFgacgatgccttcagcacaAACTCTGCTCCATATTCC 4564 A446G ER CTCCATCATCCTTTTACAC4564 A446G AR gggacggtcggtagatACGGCTCCTATTCCCAGT 4564 A446G GRgctggctcggtcaagaACGGCTCCTATTCCCAGC 5569 A605G ERgacgatgccttcagcacaCCTCTGTTCCTTCCCTCT 5569 A605G EF AGTGTGGTCTCCGAATGT5569 A605G AF gggacggtcggtagatTGGAGCATGGGAGCCACA 5569 A605G GFgctggctcggtcaagaTGGAGCATGGGAGCCACG 6872 A254G EF CATCAAGGCAGACCAA 6872A254G ER GAAGGAGAGCAAAGGG 6872 A254G AFgggacggtcggtagatAAGATACCTAAATAACAA 6872 A254G GFgctggctcggtcaagaAAGATACCTAAATAACAG 8164 A251T EF CACAAAATACACCAACAA 8164A251T ER CATTGATAAGGAATAAGGA 8164 A251T AFgggacggtcggtagatACATACGCACAAAAATTA 8164 A251T TFgctggctcggtcaagaACATACGCACAAAAATTT 8242 A251G EF AGACCCACATTCACACAC 8242A251G ER TTACACGTCAGCTTCCTC 8242 A251G ARgggacggtcggtagatTTACACACACAGTTAGAT 8242 A251G GRgctggctcggtcaagaTTACACACACAGTTAGAC 8589 C378T EFgacgatgccttcagcacaGTTGTTTGGTTTGTTGTTTT 8589 C378T ER GCTTGGCTTCCTATGTCT8589 C378T CR gggacggtcggtagatTAGCATCAATGCTGGGAG 8589 C378T TRgctggctcggtcaagaTAGCATCAATGCTGGGAA 10771 C64G EFgacgatgccttcagcacaCCAGGGAAGAGCAGAACC 10771 C64G ER TGTACGGGAAGAGGCAGA10771 C64G CR gggacggtcggtagatAGGGTGACACAGGCCACG 10771 C64G GRgctggctcggtcaagaAGGGTGACACAGGCCACC 12399 A55G EFgacgatgccttcagcacaGTGTGTTTCGCAGGAGGA 12399 A55G ER AGTTTCTCTGGCTGGTGTTG12399 A55G AR gggacggtcggtagatTAGGGGGCTGCCAGGCTT 12399 AS5G GRgctggctcggtcaagaTAGGGGGCTGCCAGGCTC

TABLE 2b Oligonucleotide primers used for genotyping usingPyrosequencing The baySNP number refers to an internal numbering of thePA SNPs. Primer sequences are listed for preamplification of the genomicfragments and for sequencing of the SNP using the pyrosequencing method.Bio: Bio- tinylated Oligonucleotide. baySNP Name Sequence 7372 Primer FGTGGAGCGGGAGCGAAC 7372 Primer R Bio-CCCCTCAAACCGTCAG 7372 Seq.GGGCATTCTCAGTGG 900066 Primer F BIO-TGCCGGGAACGTGGACTAGA 900066 Primer RCCGGCCTCTGTTTATGTAGTTCA 900066 Seq. CTTCCCCCGCCCGGGCCCGCC 900073 PrimerF BIO-GGCCCCGGCTCCACGTGCTTTC 900073 Primer R TGAGAACCGGCTCTGTTGGTGCG900073 Seq. CTGTGCTCTCCCTCCTCCCC

TABLE 2d Oligonucleotide primers used for genotyping using TaqMan ThebaySNP number refers to an internal numbering of the PA SNPs. Primersequences are listed for amplification of the genomic fragments. Inaddition the respective fluo- rescent hybridisation probes are listed.If not otherwise stated, all fluorescent probes have a ‘minor groovebinder’ (MGB) attached (Kutyavin et al., Nucleic Acids Research 2000,28:655-661). baySNP F-Sequence R-Sequence VIC-MGB FAM-MGB  1278ACCGAGCTGGAGGGGAGTT CACAGCCTGGCCACCTAACA CAGAGCAAGCTaAGGAAGAGCAAGCTgAGGAG 10771 CTGGGCCCACCGAGTTAC GATCTCTGTGAGTGTGCGTCTGTAGGAAGcGTGGCCT CAAGGAAGgGTGGC

TABLE 3 PA SNPs, SNP classes and putative PA genes The baySNP numberrefers to an internal numbering of the PA SNPs. Listed are the differentpolymorphisms found in our association study. Also from the associationstudy we defined SNP classes; with ADR being adverse drug reactionrelated, with EFF being drug efficacy related and CVD beingcardiovascular disease related. ADR3 and ADR5 relate to advanced andsevere ADR, whereas VEFF and UEFF relate to very high/low and ultrahigh/low drug efficacy (see table 1b). Also accession numbers anddescriptions of those gene loci are given that are most homologous tothe PA genes as listed in the sequences section (see below). Homologousgenes and their accession numbers could be found by those skilled in theart in the Genbank database. BAYSNP SNP class GTYPE11 GTYPE12 GTYPE22NCBI DESCRIPTION 160 ADR5 CC CT TT HS34804 Human thermostable phenolsulfotransferase (STP2) gene, partial cds. 1278 CVD AA AG GG M23442Human interleukin 4 (IL-4) gene, complete cds. 1371 CVD CC CT TT U41078Human membrane-type matrix metalloproteinase-1 mRNA, complete cds. 1806CVD AA AG GG AF106202 Homo sapiens endothelial cell protein C receptorprecursor (EPCR) gene, complete cds. 2178 CVD CC CT TT D25303 Human mRNAfor integrin alpha subunit, complete cds. 2198 CVD CC CT TT L07555 Homosapiens early activation antigen CD69 mRNA, complete cds. 2214 CVD AA ATTT M14660 Human ISG-54K gene (interferon stimulated gene) encoding a 54kDA protein, exon 2. 2267 CVD CC CT TT M13008 Human granulocytecolony-stimulating factor gene. 3907 ADR5 AA AC CC HSCHK2 H. sapiens HK2mRNA for hexokinase II 4564 ADR3 AA AG GG HSOXA1HS H. sapiens OXA1HsmRNA 4869 CVD AA AG GG D83646 Homo sapiens mRNA for metalloproteinase,complete cds. 5569 ADR3 AA AG GG HSBCL2C Human bcl-2 mRNA. 6872 CVD AGGG null ABCG2 ABCG2: ATP-binding cassette, sub-family G (WHITE), member2 7372 CVD CC CT TT M16006 Human plasminogen activator inhibitor-1(PAI-1) mRNA, complete cds. 8164 CVD AA AT TT NM_000927 ABCB1:ATP-binding cassette, sub-family B (MDR/TAP), member 1 8242 CVD AA AG GGAF165281 Homo sapiens ATP cassette binding transporter 1 (ABC1) mRNA,complete cds. 8589 VEFF CC CT TT HSMDMCSF Humanmethylenetetrahydrofolate dehydrogenase- methenyltetrahydrofolatecyclohydrolase-formyltetrahydrofolate synthetase mRNA, complete cds.10771 CVD CC CG GG D37932 Human mRNA for HPC-1 (STX1A: syntaxin 1A(brain)) 12399 ADR5 AA AG GG D86982 Human mRNA for KIAA0229 gene,partial cds. 900066 CVD CC CT TT AF275948 ABCA1 (SNP HTM 2) 900073 CVDCC CG GG AF275948 ABCA1 —(SNP HTM 15)Null: not defined.

TABLE 4 Cohorts Given are names (as used in table 5) and formations ofthe various cohorts that were used for genotyping COHORT DefinitionHELD_ALL_GOOD/BAD Healthy elderly individuals of both genders with goodor bad serum lipid profiles (as defined in table 1a) HELD_FEM_GOOD/BADHealthy elderly individuals (female) with good or bad serum lipidprofiles (as defined in table 1a) HELD_MAL_GOOD/BAD Healthy elderlyindividuals (male) with good or bad serum lipid profiles (as defined intable 1a) CVD_ALL_CASE/CTRL Individuals with diagnosis of cardiovasculardisease and healthy controls (both genders) CVD_FEM_CASE/CTRLIndividuals with diagnosis of cardiovascular disease and healthycontrols (female) CVD_MAL_CASE/CTRL Individuals with diagnosis ofcardiovascular disease and healthy controls (male) HELD_FEM_ADRCTRLFemale individuals that tolerate adminstration of cerivastatin withoutexhibiting signs of ADR (as defined in table 1b) HELD_FEM_ADRCASE Femaleindividuals that exhibited ADR (as defined in table 1b) uponadministration of cerivastatin HELD_MAL_ADRCTRL Male individuals thattolerate adminstration of cerivastatin without exhibiting signs of ADR(as defined in table 1b) HELD_MAL_ADRCASE Male individuals thatexhibited ADR (as defined in table 1b) upon administration ofcerivastatin HELD_ALL_ADRCTRL Individuals of both genders that tolerateadminstration of cerivastatin without exhibiting signs of ADR (asdefined in table 1b) HELD_ALL_ADRCASE Individuals of both genders thatexhibited ADR (as defined in table 1b) upon administration ofcerivastatin HELD_FEM_LORESP Female individuals with a minor response tocerivastatin administration (as defined in table 1b) HELD_FEM_HIRESPFemale individuals with a high response to to cerivastatinadministration (as defined in table 1b) HELD_FEM_HIHDL/LOHDL Healthyelderly individuals (female) with high or low serum HDL cholesterollevels (as defined in table 1c) HELD_MAL_HIHDL/LOHDL Healthy elderlyindividuals (male) with high or low serum HDL cholesterol levels (asdefined in table 1c) HELD_ALL_HIHDL/LOHDL Healthy elderly individuals ofboth genders with high or low serum HDL cholesterol levels (as definedin table 1c) HELD_FEM_ADR3CASE Female individuals that exhibitedadvanced ADR (as defined in table 1b) upon administration ofcerivastatin HELD_MAL_ADR3CASE Male individuals that exhibited advancedADR (as defined in table 1b) upon administration of cerivastatinHELD_ALL_ADR3CASE Individuals of both genders that exhibited advancedADR (as defined in table 1b) upon administration of cerivastatinHELD_FEM_VLORESP Female individuals with a very low response tocerivastatin administration (as defined in table 1b) HELD_FEM_VHIRESPFemale individuals with a very high response to cerivastatinadministration (as defined in table 1b) HELD_FEM_ADR5CASE Femaleindividuals that exhibited severe ADR (as defined in table 1b) uponadministration of cerivastatin HELD_MAL_ADR5CASE Male individuals thatexhibited severe ADR (as defined in table 1b) upon administration ofcerivastatin HELD_ALL_ADR5CASE Individuals of both genders thatexhibited severe ADR (as defined in table 1b) upon administration ofcerivastatin HELD_FEM_ULORESP Female individuals with a ultra lowresponse to cerivastatin administration (as defined in table 1b)HELD_FEM_UHIRESP Female individuals with a ultra high response to tocerivastatin administration (as defined in table 1b)

Table 5a and 5b Cohort sizes and p-values of PA SNPs

The baySNP number refers to an internal numbering of the PA SNPs. Cpvaldenotes the classical Pearson chi-squared test, Xpval denotes the exactversion of Pearson's chi-squared test, LRpval denotes thelikelihood-ratio chi-squared test, . . . Cpvalue, Xpvalue, and LRpvalueare calculated as described in (SAS/STAT User's Guide of the SASOnlineDoc, Version 8), (L. D. Fisher and G. van Belle, Biostatistics,Wiley Interscience 1993), and (A. Agresti, Statistical Science 7, 131(1992)). The GTYPE and Allele p values were obtained through therespective chi square tests when comparing COHORTs A and B. For GTYPE pvalue the number of patients in cohort A carrying genotypes 11, 12 or 22(FQ11 A, FQ 12 A, FQ 22 A; genotypes as defined in table 3) werecompared with the respective patients in cohort B (FQ11 B, FQ 12 B, FQ22 B; genotypes as defined in table 3) resulting in the respective chisquare test with a 3×2 matrix. For Allele p values we compared theallele count of alleles 1 and 2(A1 and A2) in cohorts A and B,respectively (chi-square test with a 2×2 matrix). SIZE A and B: Numberof patients in cohorts A and B, respectively. See table 4 for definitionof COHORTs A and B. TABLE 5a Cohort sizes and frequency of alleles andgenotypes SIZE FQ FQ FQ FQ FQ SIZE FQ FQ FQ FQ FQ baySNP A1 A2 COHORT AA 1A 2A 11A 12A 22A COHORT B B 1B 2B 11B 12B 22B 160 C T HELD_MAL_(—) 912 6 3 6 0 HELD_MAL_(—) 72 56 88 10 36 26 ADRCASE5ULN ADRCTRL 160 C THELD_ALL_(—) 27 28 26 7 14 6 HELD_ALL_(—) 155 117 193 20 77 58ADRCASE5ULN ADRCTRL 1278 A G HELD_ALL_CASE 45 12 78 2 8 35 HELD_ALL_(—)40 3 77 0 3 37 CTRL 1278 A G HELD_MAL_CASE 14 3 25 1 1 12 HELD_MAL_(—)18 36 0 0 0 18 CTRL 1371 C T CVD_MAL_CASE 66 73 59 20 33 13 CVD_MAL_(—)34 26 42 5 16 13 CTRL 1806 A G HELD_MAL_CASE 14 23 5 9 5 0 HELD_MAL_(—)17 33 1 16 1 0 CTRL 1806 A G CVD_FEM_CASE 35 63 7 30 3 2 CVD_FEM_(—) 4070 10 30 10 0 CTRL 2178 C T CVD_MAL_CASE 69 49 89 9 31 29 CVD_MAL_(—) 3435 33 10 15 9 CTRL 2198 C T CVD_FEM_CASE 35 13 57 2 9 24 CVD_FEM_(—) 3927 51 6 15 18 CTRL 2214 A T HELD_MAL_CASE 14 15 13 3 9 2 HELD_MAL_(—) 1811 25 0 11 7 CTRL 2267 C T HELD_FEM_CASE 31 55 7 24 7 0 HELD_FEM_(—) 2243 1 21 1 0 CTRL 3907 A C HELD_MAL_(—) 9 14 4 6 2 1 HELD_MAL_(—) 70 11327 43 27 0 ADRCASE5ULN ADRCTRL 4564 A C HELD_MAL_(—) 24 37 11 13 11 0HELD_MAL_(—) 72 127 17 55 17 0 ADRCASE3ULN ADRCTRL 4869 A G CVD_MAL_CASE37 7 67 2 3 32 CVD_MAL_(—) 20 6 34 0 6 14 CTRL 5569 A G HELD_MAL_(—) 246 42 0 6 18 HELD_MAL_(—) 65 37 93 4 29 32 ADRCASE3ULN ADRCTRL 5569 A CHELD_ALL_(—) 59 22 96 2 18 39 HELD_ALL_(—) 145 82 208 12 58 75ADRCASE3ULN ADRCTRL 6872 A C HELD_FEM_CASE 31 25 37 25 6 0 HELD_FEM_(—)22 22 22 22 0 0 CTRL 7372 C T CVD_FEM_CASE 33 36 30 3 30 0 CVD_FEM_(—)40 50 30 12 26 2 CTRL 8164 A T HELD_MAL_BAD 20 18 22 4 10 6 HELD_MAL_(—)37 48 26 16 16 5 GOOD 8242 A C HELD_MAL_CASE 13 12 14 0 12 1HELD_MAL_(—) 18 22 14 4 14 0 CTRL 8589 C T HELD_FEM_(—) 148 254 42 11034 4 HELD_FEM_(—) 133 208 58 81 46 6 VHIRESP VLORESP 10771 C CHELD_MAL_BAD2 309 366 252 117 132 60 HELD_MAL_(—) 348 451 245 147 157 44GOOD2 12399 A G HELD_FEM_(—) 18 36 0 18 0 0 HELD_FEM_(—) 82 147 17 67 132 ADRCASE5ULN ADRCTRL 900066 C T HELD_MAL_BAD 18 32 4 16 0 2HELD_MAL_(—) 33 55 11 24 7 2 GOOD 900066 C T HELD_MAL_CASE 8 11 5 5 1 2HELD_MAL_(—) 14 26 2 12 2 0 CTRL 900073 C C HELD_MAL_BAD 12 4 20 0 4 8HELD_MAL_(—) 32 25 39 3 19 10 GOOD 900073 C G CVD_MAL_CASE 69 47 91 9 2931 CVD_MAL_(—) 32 12 52 1 10 21 CTRL

TABLE 5b p-values of PA SNPs. A SNP is considered as associated tocardiovascular disease, adverse statin response or to efficacy of statintreatment, respectively, when one of the p values is equal or below0.05. GTYPE GTYPE GTYPE ALLELE ALLELE ALLELE BAYSNP COMPARISON CPVALXPVAL LRPVAL CPVAL XPVAL LRPVAL 160 HELD_MAL_ADR5ULN 0.0619 0.08830.0182 0.0244 0.0403 0.0251 160 HELD_ALL_ADR5ULN 0.1249 0.128 0.13830.0506 0.0699 0.0529 1278 HELD_ALL_CC 0.1321 0.1284 0.0866 0.0279 0.03190.0226 1278 HELD_MAL_CC 0.2537 0.1835 0.1756 0.0443 0.0786 0.0232 1371CVD_MAL 0.0747 0.0799 0.0729 0.0222 0.0255 0.0217 1806 HELD_MAL_CC0.0364 0.0671 0.0318 0.048 0.0824 0.0423 1806 CVD_FEM 0.0652 0.06 0.04040.6299 0.7973 0.6288 2178 CVD_MAL 0.0905 0.0909 0.0964 0.0283 0.03490.0289 2198 CVD_FEM 0.1254 0.1443 0.1193 0.0282 0.0408 0.0268 2214HELD_MAL_CC 0.0619 0.0799 0.0334 0.0629 0.0769 0.0625 2267 HELD_FEM_CC0.0707 0.1196 0.0536 0.0833 0.1357 0.0627 3907 HELD_MAL_ADR5ULN 0.0150.0811 0.0803 0.7678 1 0.7708 4564 HELD_MAL_ADR3ULN 0.0381 0.0673 0.04310.0589 0.0956 0.0696 4869 CVD_MAL 0.0653 0.0631 0.0529 0.3744 0.53810.3823 5569 HELD_MAL_ADR3ULN 0.0719 0.0715 0.0418 0.0273 0.0302 0.02055569 HELD_ALL_ADR3ULN 0.1337 0.1242 0.1207 0.043 0.0455 0.0387 6872HELD_FEM_CC 0.0284 0.0709 0.0083 0.323 0.4276 0.3232 7372 CVD_FEM 0.0290.0188 0.0168 0.3309 0.3986 0.3311 8164 HELD_MAL_LIP 0.1383 0.1453 0.1320.0403 0.0483 0.0407 8242 HELD_MAL_CC 0.1073 0.0695 0.0434 0.2429 0.30460.2426 8589 HELD_FEM_VEFF 0.0545 0.0568 0.0542 0.0184 0.0204 0.018410771 HELD_MAL_LIP2 0.0567 0.0565 0.0568 0.0375 0.0402 0.0376 12399HELD_FEM_ADR5ULN 0.1442 0.1446 0.0386 0.0434 0.0464 0.0078 900066HELD_MAL_LIP 0.101 0.1043 0.0328 0.449 0.5653 0.4405 900066 HELD_MAL_CC0.1446 0.1554 0.1094 0.0355 0.0801 0.0387 900073 HELD_MAL_LIP 0.08490.0879 0.0614 0.0465 0.0732 0.0382 900073 CVD_MAL 0.0978 0.0962 0.07870.026 0.0306 0.0224

TABLE 6a Correlation of genotypes of PA SNPs to relative risk Fordiagnostic conclusions to be drawn from genotyping a particular patientwe calculated the relative risk RR1, RR2, RR3 for the three possiblegenotypes of each SNP. Given the genotype frequencies as gtype1 gtype2gtype3 case N11 N12 N13 control N21 N22 N23 we calculate${{RR}\quad 1} = {\frac{N\quad 11}{N\quad 21}/\frac{{N\quad 12} + {N\quad 13}}{{N\quad 22} + {N\quad 23}}}$${{RR}\quad 2} = {\frac{N\quad 12}{N\quad 22}/\frac{{N\quad 11} + {N\quad 13}}{{N\quad 21} + {N\quad 23}}}$${{RR}\quad 3} = {\frac{N\quad 13}{N\quad 23}/\frac{{N\quad 11} + {N\quad 12}}{{N\quad 21} + {N\quad 22}}}$

Here, the case and control populations represent any case-control-grouppair, or bad(case)-good(control)-group pair, respectively (due to theirincreased response to statins, ‘high responders’ are treated as a casecohort, whereas ‘low responders’ are treated as the respective controlcohort). A value RR1>1, RR2>1, and RR3>1 indicates an increased risk forindividuals carrying genotype 1, genotype 2, and genotype 3,respectively. For example, RR1=3 indicates a 3-fold risk of anindividual carrying genotype 1 as compared to individuals carryinggenotype 2 or 3 (a detailed description of relative risk calculation andstatistics can be found in (Biostatistics, L. D. Fisher and G. vanBelle, Wiley Interscience 1993)). The baySNP number refers to aninternal numbering of the PA SNPs and can be found in the sequencelisting. null: not defined.

In cases where a relative risk is not given in the table (three timeszero or null) the informative genotype can be drawn from the right partof the table where the frequencies of genotypes are given in the casesand control cohorts. For example BaySNP 12399 gave the followingresults: BAYSNP COMPARISON GTYPE1 GTYPE2 GTYPE3 RR1 RR2 RR3 12399HELD_FEM_ADR5ULN AA AG GG null 0 0 FQ1_A FQ2_A FQ3_A FQ1_B FQ2_B FQ3_B18 0 0 67 13 2

It can be concluded that a AG or GG genotype is only present in thecontrol cohort; these genotypes are somehow protective against ADR. Ananalogous proceeding can be used to determine protective alleles if norelative risk is given (table 6b). BAYSNP COMPARISON GTYPE1 GTYPE2GTYPE3 RR1 RR2 RR3 FQ1_A FQ2_A FQ3_A FQ1_B FQ2_B FQ3_B 160 HELD_MAL_(—)CC CT TT 2.62 1.86 0 3 6 0 10 36 26 ADR5ULN 160 HELD_ALL_(—) CC CT TT2.01 1.08 0.53 7 14 6 20 77 58 ADR5ULN 1278 HELD_ALL_CC AA AG GG 1.931.45 0.63 2 8 35 0 3 37 1278 HELD_MAL_CC AA AG GG 2.38 2.38 0.4 1 1 12 00 18 1371 CVD_MAL CC CT TT 1.3 1.04 0.7 20 33 13 5 16 13 1806HELD_MAL_CC AA AG GG 0.43 2.31 null 9 5 0 16 1 0 1806 CVD_FEM AA AG GG1.5 0.45 2.21 30 3 2 30 10 0 2178 CVD_MAL CC CT TT 0.66 1.01 1.24 9 3129 10 15 9 2198 CVD_FEM CC CT TT 0.5 0.72 1.66 2 9 24 6 15 18 2214HELD_MAL_CC AA AT TT 2.64 1.08 0.43 3 9 2 0 11 7 2267 HELD_FEM_CC CC CTTT 0.61 1.64 null 24 7 0 21 1 0 3907 HELD_MAL_(—) AA AC CC 1.22 0.499.75 6 2 1 43 27 0 ADR5ULN 4564 HELD_MAL_(—) AA AG GG 0.49 2.05 null 1311 0 55 17 0 ADR3ULN 4869 CVD_MAL AA AG GG 1.57 0.47 1.53 2 3 32 0 6 145569 HELD_MAL_(—) AA AG GG 0 0.51 2.34 0 6 18 4 29 32 ADR3ULN 5569HELD_ALL_(—) AA AG GG 0.48 0.74 1.54 2 18 39 12 58 75 ADR3ULN 6872HELD_FEM_CC AG GG null 0.53 1.88 null 25 6 0 22 0 0 7372 CVD_FEM CC CTTT 0.39 3.04 0 3 30 0 12 26 2 8164 HELD_MAL_LIP AA AT TT 0.46 1.19 1.794 10 6 16 16 5 8242 HELD_MAL_CC AA AG GG 0 2.31 2.5 0 12 1 4 14 0 8589HELD_FEM_VEFF CC CT TT 1.36 0.75 0.75 110 34 4 81 46 6 10771HELD_MAL_LIP2 CC CG GG 0.91 0.95 1.28 117 132 60 147 157 44 12399HELD_FEM_(—) AA AG GG null 0 0 18 0 0 67 13 2 ADR5ULN 900066HELD_MAL_LIP CC CT TT 2.2 0 1.47 16 0 2 24 7 2 900066 HELD_MAL_CC CC CTTT 0.49 0.9 3.33 5 1 2 12 2 0 900073 HELD_MAL_LIP CC CG GG 0 0.46 2.89 04 8 3 19 10 900073 CVD_MAL CC CG GG 1.37 1.15 0.77 9 29 31 1 10 21

TABLE 6b Correlation of PA SNP alleles to relative risk For diagnosticconclusions to be drawn from genotyping a particular patient wecalculated the relative risks RR1, and RR2 for the two possible allelesof each SNP. Given the allele frequencies as allele1 allele2 case N11N12 control N21 N22 we calculate${{RR}\quad 1} = {\frac{N\quad 11}{N\quad 21}/\frac{N\quad 12}{N\quad 22}}$${{RR}\quad 2} = {\frac{N\quad 12}{N\quad 22}/\frac{N\quad 11}{N\quad 21}}$

Here, the case and control populations represent any case-control-grouppair, or bad(case)-good(control)-group pair, respectively (due to theirincreased response to statins, ‘high responders’ are treated as a casecohort, whereas ‘low responders’ are treated as the respective controlcohort). A value RR1>1, and RR2>1 indicates an increased risk forindividuals carrying allele 1, and allele 2, respectively. For example,RR1=3 indicates a 3-fold risk of an individual carrying allele 1 ascompared to individuals not carrying allele 1 (a detailed description ofrelative risk calculation and statistics can be found in (Biostatistics,L. D. Fisher and G. van Belle, Wiley Interscience 1993)). The baySNPnumber refers to an internal numbering of the PA SNPs and can be foundin the sequence listing. null: not defined. BAYSNP ALLELE1 ALLELE2COMPARISON RR1 RR2 SIZE A FREQ1 A FREQ2 A SIZE B FREQ1 B FREQ2 B 160 C THELD_MAL_ADR5ULN 2.76 0.36 9 12 6 72 56 88 160 C T HELD_ALL_ADR5ULN 1.630.61 27 28 26 155 117 193 1278 A G HELD_ALL_CC 1.59 0.63 45 12 78 40 377 1278 A G HELD_MAL_CC 0.08 13 14 3 25 18 36 0 1371 C T CVD_MAL 1.260.79 66 73 59 34 26 42 1806 A G HELD_MAL_CC 0.49 2.03 14 23 5 17 33 11806 A G CVD_FEM 1.15 0.87 35 63 7 40 70 10 2178 C T CVD_MAL 0.8 1.25 6949 89 34 35 33 2198 C T CVD_FEM 0.62 1.62 35 13 57 39 27 51 2214 A THELD_MAL_CC 1.69 0.59 14 15 13 18 11 25 2267 C T HELD_FEM_CC 0.64 1.5631 55 7 22 43 1 3907 A C HELD_MAL_ADR5ULN 0.85 1.17 9 14 4 70 113 274564 A G HELD_MAL_ADR3ULN 0.57 1.74 24 37 11 72 127 17 4869 A G CVD_MAL0.81 1.23 37 7 67 20 6 34 5569 A G HELD_MAL_ADR3ULN 0.45 2.23 24 6 42 6537 93 5569 A G HELD_ALL_ADR3ULN 0.67 1.49 59 22 96 145 82 208 6872 A GHELD_FEM_CC 0.85 1.18 31 25 37 22 22 22 7372 C T CVD_FEM 0.84 1.19 33 3630 40 50 30 8164 A T HELD_MAL_LIP 0.6 1.68 20 18 22 37 48 26 8242 A GHELD_MAL_CC 0.71 1.42 13 12 14 18 22 14 8589 C T HELD_FEM_VEFF 1.31 0.76148 254 42 133 208 58 10771 C G HELD_MAL_LIP2 0.88 1.13 309 366 252 348451 245 12399 A G HELD_FEM_ADR5ULN null 0 18 36 0 82 147 17 900066 C THELD_MAL_LIP 1.38 0.73 18 32 4 33 55 11 900066 C T HELD_MAL_CC 0.42 2.48 11 5 14 26 2 900073 C G HELD_MAL_LIP 0.41 2.46 12 4 20 32 25 39 900073C G CVD_MAL 1.25 0.8 69 47 91 32 12 52

1. An isolated polynucleotide encoded by a phenotype associated (PA)gene; the polynucleotide is selected from the group comprising SEQ ID 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21with allelic variation as indicated in the sequences section containedin a functional surrounding like full length cDNA for PA genepolypeptide and with or without the PA gene promoter sequence.
 2. Anexpression vector containing one or more of the polynucleotides ofclaim
 1. 3. A host cell containing the expression vector of claim
 2. 4.A substantially purified PA gene polypeptide encoded by a polynucleotideof claim
 1. 5. A method for producing a PA gene polypeptide, wherein themethod comprises the following steps: a) culturing the host cell ofclaim 3 under conditions suitable for the expression of the PA genepolypeptide; and b) recovering the PA gene polypeptide from the hostcell culture.
 6. A method for the detection of a polynucleotide of claim1 or a PA gene polypeptide of claim 4 comprising the steps of:contacting a biological sample with a reagent which specificallyinteracts with the polynucleotide or the PA gene polypeptide.
 7. Amethod of screening for agents which regulate the activity of a PA genecomprising the steps of:# contacting a test compound with a PA genepolypeptide encoded by any polynucleotide of claim 1; and detecting PAgene activity of the polypeptide, wherein a test compound whichincreases the PA gene polypeptide activity is identified as a potentialtherapeutic agent for increasing the activity of the PA gene polypeptideand wherein a test compound which decreases the PA activity of thepolypeptide is identified as a potential therapeutic agent fordecreasing the activity of the PA gene polypeptide.
 8. A reagent thatmodulates the activity of a PA polypeptide or a polynucleotide whereinsaid reagent is identified by the method of the claim
 7. 9. Apharmaceutical composition, comprising: the expression vector of claim 2or the reagent of claim 8 and a pharmaceutically acceptable carrier. 10.Use of the reagent according to claim 8 for the preparation of amedicament.
 11. A method for determining whether a human subject has, oris at risk of developing a cardiovascular disease, comprisingdetermining the identity of nucleotide variations as indicated in thesequences section of SEQ ID 1-21 of the PA gene locus of the subject andwhere the SNP class of the SNP is “CVD” as can be seen from table 3;whereas a “risk” genotype has a risk ratio of greater than 1 as can beseen from table
 6. 12. A method for determining a patient's individualresponse to statin therapy, including drug efficacy and adverse drugreactions, comprising determining the identity of nucleotide variationsas indicated in the sequences section of SEQ ID 1-21 of the PA genelocus of the subject and where the SNP class of the SNP is “ADR”, “EFF”or both as can be seen from table 3; whereas the probability for suchresponse can be seen from table
 6. 13. Use of the method according toclaim 12 for the preparation of a medicament tailored to suit apatient's individual response to statin therapy.
 14. A kit for assessingcardiovascular status or statin response, said kit comprising a)sequence determination primers and b) sequence determination reagents,wherein said primers are selected from the group comprising primers thathybridize to polymorphic positions in human PA genes according to claim1; and primers that hybridize immediately adjacent to polymorphicpositions in human PA genes according to claim
 1. 15. A kit as definedin claims 12 detecting a combination of two or more, up to all,polymorphic sites selected from the groups of sequences as defined inclaim
 1. 16. A kit for assessing cardiovascular status or statinresponse, said kit comprising one or more antibodies specific for apolymorphic position defined in claim 1 within the human PA genepolypeptides and combinations of any of the foregoing.